Then and Now

Then and Now use the following form to describe your dally routines before you were a college student versus a typical days routine now that you are a college student. Using the form as a reference, answer the questions that follow in at least 50 words each.Daily Routine Form Day Before college After college Sunday Wake up, eat, take care of the kids, fix around and just relaxed up, eat, take care of the kids, watch movies with them,and Just relax, and sometimes make sure I did finish all my school work Monday Wake up, fix breakfast for the kids get my aught ready for school drop her off, go to work am-pm, pick up my daughter, get back home, feed the kids, play with them, watch TV, fix dinner, get them ready for bed Wake up, fix breakfast for the kids and me, take my daughter to school do 2 hours of school work, flu lunch get dress go to work 3-11 pm get back home make sure the kids are k and sleeping do 1 hour of school watch TV and go to sleep Tuesday Wake up, fix breakfast for the kids get my daughter ready for school drop her off, go to work am-pm, pick up my daughter, get back home, feed the kids, play with them, watch v, fix dinner, get them ready for bed Wake up, fix breakfast for the kids and me, take my daughter to school do 2 hours of school work, fix lunch get dress go to work 3-11 pm get back home make sure the kids are k and sleeping do 1 hour of school watch TV and go to sleep Wednesday Wake up, fix breakfast for the kids get my daughter ready for school drop her off, pick up my daughter, get back home, feed the kids, play with them, watch TV, fix dinner, get them ready for bed Wake up, fix breakfast for the kids and me, take my daughter to school do 2 hours of school work, ix lunch get dress go to work 3-11 pm get back home make sure the kids are k and sleeping do 1 hour of school watch TV and go to sleep Thursday Wake up, fix breakfast for the kids get my daughter ready for school drop her off, go to work take my daughter to school, fix lunch, p ick my daughter up from school, feed the kids, play with them, fix dinner, get them ready for bed.Friday Wake up, fix breakfast for the kids get my daughter ready for school drop her off, go to work am-pm, pick up my daughter, get back home, feed the kids, play with them, watch TV, fix dinner, get hem ready for bed Wake up, fix breakfast for the kids and me, take my daughter to school do 2 hours of school work, fix lunch get dress go to work 3-11 pm get back home make sure the kids are k and sleeping do 1 hour of school watch TV and go to sleep Saturday Wake up, fix breakfast for the kids, go to work am-pm, get back home, feed the kids, play with them, watch TV, fix dinner, get them ready for bed Wake feed the kids, play with them, fix dinner, get them ready for bed. Daily Routine Questions 1. What are the major differences in your daily routine now that you are in school? The major differences in my daily routine now that IM in school and before are.When I wasn't in school I use to work the morning shift, and after work I had pretty much the rest of the day to Just cook and play with the kids or Just waste time, now I work the night shift 3-11 pm I use my morning time to make sure the kids are taken care of and as part of my routine I incorporated time for school in the morning as well as at nights after I get back from work 2. Do you have an effective balance in the use of your time and your priorities? Why or why not? I believe I have a pretty effective balance on the used of my time and my priorities cause so far so good , I haven't had any problems or issues Just yet, I always been to turn my assignments on time, and be able to expend time with my kids, and not neglect them anything 3. Describe your biggest obstacle to completing projects or assignments and how you will overcome it? Although o have a pretty effective balance of my time I would have to say my biggest obstacle to complete projects or assignments on time would be managing time.Up until now V ive been pretty good at it, but having two kids, working full time and being back in school can get a little crazy and hectic especially when my kids get sick, I'll just have to have a backup plan for unexpected events such as them getting sick or me getting sick. 4. What are some time-management strategies you have learned this week that you can implement immediately? How will you use them? Some of the time management strategies I have learn this week is keeping a time log, this is a helpful way to determine how you are using your time, use a planning tool, get organized and schedule your time appropriately by Develop blocks of study time effectively manage my time by organizing and proportioning tasks such as schoolwork, Then and Now Then and Now use the following form to describe your daily routines before you were a college student versus a typical days routine now that you are a college student. Using the form as a reference, answer the questions that follow in at least 50 words each. Dally Routine Form Day Before college After college Sunday I would lounge around In my pajamas all day and Just watch TV with my sister and family. I get up every morning and check on the progression of my assignments that re usually due and I work to complete them as well as make sure I have posted for participation for the week.I also spend quality time with my husband, lounging around and watching TV and playing video games. Monday I would go to the lake and exercise (Running) then go home and spend the day doing various things ranging from watching TV to cooking dinner to doing laundry and help my sister with her homework. I usually get up before 5 to help my husband to get ready for work then send him off. Then I am up clean ing the house and completing horses as well as running errands for the household, and my husband as well as looking at my assignments fir the week and writing them down in my planner and posting reminders in my phone and computer.I also check my grades for the week and have check ins with my enrollment advisor, Ashley, and we go over my progress in my courses and she gives feedback on my grades and what I can and should do better In regards to my grades. Tuesday I would wake up and exercise In a different location such as the forest behind my house. Then I would run around all day with my aunt and uncle getting things done such as paying bills, grocery shopping, and searching for schools for me to go to.I basically do the same thing I do Monday, but I am also making lists of things my husband and I need for the household, work, and school. I am also checking on my school work and completing my chapter readings for my courses. Wednesday I usually get up and exercise that morning, the n I lounge around afterwards until 6 p. M. And then I attend bible study with my aunt and sister until 9 p. M. Then I am In bed by midnight. I am up with my husband before 6 a. M. Making sure he is ready for work then I send him off. I usually try to relax on these days and rest my body.Thursday I stay in bed on these days and just sleep or eat all day. Just like the days before I am up with my husband before 6 a. M. And helping him to shopping for shoes or anything that catches my eye. Friday I am up early to get in a workout before 8 a. M. Then I am relaxing and doing school searches and making phone calls asking about various schools and programs. These days are decided on whether or not my husband has it off. If he has the day off we are traveling the Island and exploring and eating out and going to the beach. If he does not have the day off we are up before 6 a. . Getting our day started. Saturday I wake up and go spend the day with my family playing games and making meals toge ther. I sleep in and spend the day in bed with my husband. Daily Routine Questions 1 . What are the major differences in your daily routine now that you are in school? I spend more time helping my husband get ready for work and making sure that he has everything he needs for the day. I am also checking in with my enrollment advisor and teachers to make sure I am where I need to be in school and that I am in rack with assignments and participation.I also noticed that I start my days around or before 6 a. M. And I am constantly running errands or doing chores to make sure things are done for the household, my husband, and myself. 2. Do you have an effective balance in the use of your time and your priorities? Why or why not? Yes I do. Now that I am a wife and college student at the same time, I make sure that I know ahead of time what needs to be done for my household and husband as well as what assignments are due, what they consist of, and what course they are for.I also make sure t o plan out what days I am doing certain things and what days I can afford to take time for myself and Just relax. 3. Describe your biggest obstacle to completing projects or assignments and how you will overcome it? My biggest obstacle that hinders me from completing projects or assignments would have to be time management and procrastination. I can plan out my days accordingly but sometimes I can get very busy with certain things and become side tracked. I now try to keep track of time and make sure that I am in the right place at the right time to omelet my tasks.I usually procrastinate when I am feeling lazy or I am under the weather. When this happens I will push myself to get up and get it done because it has to be done. 4. What are some time-management strategies you have learned this week that you can implement immediately? How will you use them? I learned to plan accordingly and make sure that you do not overwhelm yourself ahead of time and make sure that I have time between each task so that I can rest and give my brain a break as well as not doing more than 3 tasks a day so that I can remain stress free.

Abortion: Two Sides of the Argument

In today's society every time a person turns on the television, goes on a computer, turns on a radio, passes a billboard, or picks up a newspaper that person is bombarded with debates. There are many different debates and each debate with two parties both of which have two very different and sometimes conversational opinions. Everywhere a person looks they are faced with arguments such as president Obama's affordable healthcare act, gay rights, the matters of the death penalty being legalized or illegalized in all states, and many more. In this paper I will not talk about any of the previously stated arguments.I will however talk about one of the most heated and controversial arguments of today. Before I continue with the debate I have chosen I am stating that I will not be sharing any of my personal opinions or views on the matter. I have chosen to discuss the debate regarding abortion and the two sides of the argument. I will explain both Pro Life and Pro choice and the fallacy bot h sides use when trying to gain followers. Just about every person has an opinion or a side when it comes to the topic of abortion. I have researched percentages based on people who are Pro Life vs. Pro Choice and if abortion is ever Justified.A poll done by Gallup in May of 2013 helps to show some percentages on what side or sides the American people are taking. Based on a one hundred percent model forty five percent of people are Pro Choice, forty eight percent are Pro Life, three percent were mixed, 2 three percent did not understand the meaning of the terms provided, and two percent had no opinion. In the poll a majority of the people who participated had chosen which side of the argument they had agreed with. In another recent model done in May 2013 by Gallup shows the percentages of people's views on if abortion hould ever be legal and Justifiable.Legal under any circumstances has a vote of twenty six percent, legal under certain circumstances fifty two percent, illegal in all circumstances twenty percent, and no opinion had two percent. Pro Life is a Christian based non- profit anti- abortion organization that was founded in the year 1980 by Joseph Scheidler with the organizations head quarters in Chicago Illinois. The goal of the organization is to protect and save unborn children and their mothers with non violent protests. The organization believes that women are being exploited by the abortion industry.Also that the abortion industry is the main cause of what the organization believes is the murder of an human being. The members of Pro Life have many different tactics implemented to publically protest abortion. The Pro Life organization does not condone any violence in the protesting of abortion, but the organization does use graphic material in the â€Å"Face the Truth: Public Education Initiative†. This educational initiative uses graphic pictures of real aborted fetuses to show the aftermath of the procedure. This protest is described as à ¢â‚¬Å"to show our fellow Americans the ugly truth about abortion†.The Pro Life members line the major road ways and intersection while showcasing the graphic images. Pro Life organization also uses less graphic forms of protesting such as sidewalk counseling, youth outreach projects, public, protest, 3 presence at abortion clinics, and confronting abortionists. The majority of protesting takes place at the abortion clinics. Pro Life members will stand outside of the clinics to perform prayer vigils, last minute consulting to women and couples with abortion alternatives, pregnancy resources, as well as confidential counseling. Pro Choice I organized by the National Abortion Federation.It is the national association for professional abortion providers. The N. A. F provides training and services to qualified abortion providers in North America. The goal of the N. A. F is to ensure women's safety when to comes to abortion whether it is elective or medically necessary. Pro Choice i s centered around that women should be able to make private medical decisions with their healthcare providers and to have the right to chose what happens to their bodies. The N. A. F also supports the education for women and youth n different contraceptive methods to help prevent unplanned pregnancies.Also the organization provides resources for women to make inform decisions when it comes to their choices. The organization helps to provide women with the education about abortion, adoption, and parenthood. The organization sets quality standards for abortion care and to make sure the medical providers such as Nurses, Physician Assists, Doctors, and Mid wives are qualified to provide high quality care. N. A. F also helps with legal legislations to keep abortion legal. The organization fears what ould happen to women if abortion was to made illegal.Pro choice does protest to keep abortion legal in the United States. The Pro Choice advertisements are not as graphic as Pro Life's. Thoug h Pro Choice does have advertisements of what environments women may find themselves in if abortion were to be made illegal. Pro Life also uses education to teach women about what had happened in the 1950's to the 1960's before 4 abortion was legal in America. This is used to create awareness so that history will not repeat itself. According to Our Bodies Ourselves a Pro Choice website uses statics o show mortality rate in women to either tried to perform a self initiated abortion.Between 1950 and 1960 before women were legal able to get an abortion 1. 2 million of those women did self performed abortions. In countries where abortion is still illegal unsafe abortion is the leading cause of maternal deaths. This accounts to 219 women dying daily worldwide. Both organizations use medical facts and statics for a large part of their demonstrations, however they both employ of use of a logical fallacy to make their cases. The logical fallacy that Pro Life and Pro Choice both use is appea l to emotion. Appeal to emotion is a fallacy to manipulate the recipients' views by using an emotional trigger.This means that the parties involved may not be using actual valid intormation to persuade a person. It is very clear botn ot the organizations are against the other. The names are the first thing I had noticed. The names are completely opposite from one another. For example is a person is Pro choice then they are against life or that person is for death or murder. Also if a person is Pro Life then that implies that person is anti-choice. For another example Pro Life members eb advertisement and That I have personally seen driving on 95 is a image of a baby with the saying â€Å"Endangered species. Obviously children are not a endangered species because human beings are not endangered. This is used to evoke a strong emotional response to gain Pro Life members and not using actual facts to prove a point. Pro Choice also use the appeal to emotion to gain members as well. In a advertisement for the Pro Choice Public Education Project it contains a picture of a dirty bathroom that's covered in filth. Above what is the bathtub is a plaque that says † Operating room†. A bathroom is not an operating room this once again used to invoke emotion. The 5 emotions of despair and fear.This is used in the campaign for keeping abortion legal. Also someone the slogans each side uses appeal to emotions a well. For example Pro Life slogan â€Å"You might forgive yourself, but a dead baby sure can't†. Whether the baby to speak of is dead or alive the said baby does not have the capability to comprehend forgiveness. Both side also use TV adds to discount the other side and to persuade the viewers. The internet video 180 shows how to change a person's mind about abortion within seconds. The method that was used involved Hitler and his influence on the German people.Ray Comfort is the interviewer in the movie. Comfort asks a few young people about their feeling regarding the killing of the Jewish people. All the participants agreed that what had happened was wrong and that killing a person was wrong. Comfort then moved into abortion after using emotional response to what happened in the holocaust and comparing that to abortion. Comfort put that abortion is America's holocaust. In conclusion though both Pro Life and Pro Choice have valid points and information both of the rganizations use appeal to emotion to persuade potential followers.

Basic concepts of psychodynamic psychotherapy

In the essay, â€Å"Basic concepts of psychodynamic psychotherapy† I have delved deep into the concept psychodynamic psychotherapists. Who are they? What is their role and how they help emotionally disturbed patients. Freud was the first to formulate the concept psychodynamic psychotherapy and then with the passage of time, many types of therapies have been conceived.It is based on the simple concept that we all are emotionally related to each other and these emotions may from time to time create disturbances in our daily lives, which we are unable to find out.Therapists help us to find these problems and give the solutions. But there are certain problems too in the treatment process in the various concepts of Psychodynamic psychotherapy. These problems can arise due to transference, counter-transference, defense and resistance. All in all, this essay will be beneficial for all the people concerned and students of psychotherapy. Introduction: Psychodynamic psychotherapy involv es patients to understand their emotional turmoil and effectively deals with them.It is a therapy provided to the young adults to help them deal with the emotional problems arising out of the depression and anxiety caused due to the relationship problems either with family, peers, friends, or professors. It is a method of verbal communication enabling patients to get relief from emotional pains. People go for psychodynamic psychotherapy for number of reasons like prolonged sadness, anxiety, sexual frustration, physical symptoms without any basis, continuous feelings of isolation and loneliness, and an ardent desire to achieve more success in work and love.People ask for therapist, as they cannot solve the resolution in the time of their difficulties in their own way. The roots of the concepts psychodynamic psychotherapy had arisen out of the theories and techniques of psychoanalysis. As said by Nancy McWilliams, â€Å"Psychoanalytic therapists, including psychoanalysis, are approac hes to helping people that derive ultimately from the ideas of Sigmund Freud and his collaborators and his followers†. (McWilliams, 2004, p. 1)The overall theme of the psychodynamic approach of helping people is based on the simpler premises that the more we are honest with ourselves, the more we have chances of living a better, satisfied and useful life. Psychoanalytical and clinical writing espouses from within our unconscious level those aspects that we have not realized or are not evident and if we are aware of these disavowed aspects, we will get relief from emotional pain and also from the time and energy spent to keep ourselves at unconscious level.Michael Guy Thompson and the inheritors of Rieff argued that psychoanalysis as a field has adorned an ethic of honesty as a means to achieve therapeutic goals. Thomas Szasz in 2003 defined psychoanalysis as a â€Å"moral dialogue, not a medical treatment. † (McWilliams, 2004, p. 2) Since decades therapists have personi fied themselves as most honest in their personal analysis with the patients and also fostering the achievements as a result of the same. (McWilliams, 2004) There are differences in the goals of therapy depending on the methods of treatment that could be either expressive or supportive.Expressive therapy enables the patients to relieve themselves from symptoms through the development of awareness of feelings and thoughts. The therapy is based on the concept that difficulties, which are experienced by the adults have their emergence in childhood; children neither possess the ability of making suitable choices for themselves nor they have an independence to follow the same and the methods that are developed in the childhood are no longer effective during adulthood.With counselling, adults get to know the ineffective ways they had been adopting and today’s ways of adoption to come out of the various problems and hurdles. Another is supportive therapy, more relevant to give patien t immediate relief. Therapist adopts this approach with the previous level of functioning of a person and helps him to strengthen the ways already been adopted by him. While many patients can get benefited from one treatment but in several cases, other therapies may also be involved like family therapy, couple therapy, or group therapy, which could be separately given and also in combination.Concepts of Psychodynamic psychotherapy Psychodynamic psychotherapy provides a unique model for the mental functioning involving five key concepts, and these are: â€Å"Unconscious processes in the mental level; Transference; Counter-transference; Defense and resistance; and the past repeating itself in the present. † (Yager, Mellman & Rubin, 2005, p. 340) 1. Unconscious processes in the mental level â€Å"Unconscious is an adjectival description of areas of mental experience not available to normal awareness†. (McGrath & Margison, 2000) It is the part of the mental process about w hich we are not aware of.There could be different levels of unconscious mental activity including our inability to realize what is going on in our mind and secondly partial awareness. Most of the Freud’s writings were based on this unconscious level of mind, which is â€Å"a reservoir that contained dynamically repressed contents that were kept out of awareness because they created conflict. † (Gabbard, 2004, p. 3) Freud’s earlier attempts were his efforts to bring out to surface the unconscious part of our mind for easily identifying the problem and understanding it in a better way.Freud formulated what is known as the topographical model and the structural model. The topographical model describes the parts of the mind that functions at various levels of consciousness and creates awareness of the same. It reveals and studies the quality that is playing its part in the mental processes rather than function it is playing. On the other hand is the structural model , which delves into the three important parts of the personality i. e.id, ego and superego and they perform motivational, interactive and executive functions. In the structural model, ego is shown as different from aggressive and sexual drives. Freud explains that, â€Å"The conscious part of the ego involves that part of the mind, which performs the function of decision making, integration of perceptual data, and the mental calculation whereas the unconscious part of the ego involves defense mechanisms that are designed to counteract the power instinctual derives harbored in the id†. (Gabbard, 2004, p.4) Sexuality and aggression are drives requiring deep level defensive efforts from the ego to prevent them from becoming intrusive to the person’s functioning. According to Freud, unconsciousness continues to create an influence on our behavior even though we are unaware about it. For e. g. during one of the clinical trials, I studied the patient’s problem on his communication process. I assumed that the patient’s verbal and nonverbal communication to us was unconsciously organized, and consciously as well as unconsciously had certain meaning.This meant from his speech and non-verbal behavior, I had to find out the central conflict patient was undergoing through unwillingly organized thoughts, feelings and behaviors in his relation to the persons he was concerned. When I listened to the patient, certain portion of this conflicting tendency in his mental power was quite visible. This could be in the form of phrases, images, nonverbal behaviors etc. These signs help in interpreting the root cause of the problem. After the thorough investigation of the verbal phrase, I interpreted that his focal conflict was related to his phallic competitive wishes.But it was not clear whether his phallic conflicts were regressive arising from his struggle or he went to an extent of powerful regression towards the sadistic tendency. In other words, it w as not clear that his difficulty with phallic competitive feelings toward males had arisen from his feeling of jealousy or looking at them as rivals or his anxiety had arisen due to the sad feelings and his impulses. But one thing that I found was he often felt very anxious and often had a great feeling of anxiety over the affects and that could be created on his impulses by the people he thinks to be rivals.Psychotherapeutic acts like a friend, and as said by must act like a therapeutic distance or therapeutic neutrality; never treating with any personal desires yet always maintaining the relationship with the patient focusing on the treatment process. 2. Transference Second concept is transference, involving the relationship client feels towards his psychotherapist. It is very natural for the client to experience the feeling of transference, also known as the transference reactions. These feelings are no less than in-depth feelings of love or hate.Jacques Lacan, a psychoanalyst ex plains that this love means having a belief or faith in the other, in other words, the other person has knowledge you don’t have. (Wright, 1998) This intense belief on the part of the client can cause problem that should be solved during the process psychotherapeutic treatment. For e. g. these feelings could be mixed feelings of love and hate that can arise out of the relationship problems with parents and they look at therapist with these mixed feelings. In such a situation, there is a need to realize that psychotherapist is only trying to reduce these feelings.A patient also begins to feel that the psychotherapist has a personal ability to come out of the sense of inner worthlessness and there can be fondness and even sexual attraction with the psychotherapist. This happens as the therapeutic cure comes from the emotional feeling and removes emotional emptiness. It is said if transference is not handled carefully it can lead to disaster consequence. For e. g. many patients have their lives ruined because psychotherapists play with the patients erotic feelings in a personal way and fail to make the client understand that it is the medical treatment.In many cases transference can also make you frighten putting a stop to the treatment prematurely. For e. g. it was October 18th 2000, I had one patient in my clinic that most of the time got into the fits of anxiety and depression. I lovingly asked him several questions and during the process, I found he had an odd problem with his parents. He acknowledged the fact that his parents loved him but at the same time was always had a feeling of insecurity, anger and confusion towards his parents thinking they didn’t love him as much as they loved their other children.The first thing about him that came to my mind was he had a craving for love and it was love he needed the most. He was fourteen years old boy with smart and innocent boyish look in his face, with black and blonde hair. So my initial step of treatment started by getting emotionally close to the patient, and I initiated to give the parental care he craved for, understand his differences with his parents and try not to repeat the same mistakes what he felt his parents were doing.Slowly, his signs of depression began to reduce and he felt more relaxed and tension free. My more and more closeness with him created a situation of transference, as I soon realized he was not able to spend even few minutes of his time without calling me or having a talk with me. He was now looking at me as his saviour and parents. I soon realized this would create a more problem if I leave him, as he could feel sadder and get into more depression. I then called his parents, discussed problem with them and explained them the importance and real meaning of love.Love means not just fulfilling the responsibilities but also coming close to your child, keeping your hands on his head and saying, â€Å"I am with you. † These are magical words bes t than the medicine that can reduce the emotional pains and can trigger the self-confidence and faith in others and oneself. I gave his parents some tips to follow and soon they realized it. This was the beginning of new life for my patient as he felt more relaxed, happy and relieved from all the pains and I slowly and slowly made him realize I was only his doctor and had to go.3. Counter-transference Counter transference is a reverse of the transference. This is described for the reactions and the emotional and unconscious reactions that can be felt by psychotherapist for his client. If these feelings are taken personally then psychotherapist can get into angry bout, abusive, spiteful, indifferent, or even seductive and if the counter-transference gets very deep and intense, then psychotherapist has to stop the treatment himself and get his patient referred to someone else for client’s protection.Counter-transference should be distinguished from the feelings he generated dur ing the process of treatment, because these feeling are used for treatment. At this point we can say that feelings generated by psychotherapists could be good as well as bad as both the extremity of the emotional feelings can have adverse effect on both the psychotherapist as well as on the part of patient. With the patient I mentioned above, I also began to feel emotional closeness but I controlled my emotions and with some careful analysis of the situation and adopting the balanced approach I dealt with him.4. Defense and resistance Yet another therapeutic concept needs to be undertaken is defense and resistance. Freud defines resistance as â€Å"whatever interrupts the progress of analytic work, like getting late, missing a session, or avoiding a particular issue†. (Fink, 1997, 230) Simply defense and resistance occurs owing to the fear and fear we have to face and relinquish from the anger of the victim. In other words, the treatment task is very complex and frightening a nd there is often the fear of facing the anger of a patient and overcoming an inclination to lie to yourself.Nonetheless Lacan said resistance should be distinguished from defense, and gave the statement that â€Å"there is no other resistance to analysis than that of the analyst himself. † (Fink, 1997, p. 225) For e. g. if the psychotherapist makes interpretation or makes intervention, which seemed to be not proper clinically, the client can be defensive and that can cause interruption in the work of therapists. In other words client will only get into the process of treatment when he himself feels comfortable about. The psychotherapist must feel the awareness of the fact that to what extent of the treatment process client is willing to go.Attempts to forcefully get client deep into the treatment process without getting him emotionally prepared can result in the client getting away from the treatment itself. In my case during the initial visit of the client, he showed reluct ance in the treatment process. He often came late from the time schedule and felt hesitant in disclosing. I assured him the best of my treatment and with great patience and slight conversations slowly yet steadily made him come closer to the treatment process. Then I was comfortable with me and he too was finding comfort in the treatment.5. The past repeating itself in the present In the psychodynamic language, it implies the past experiences of the patient continue to haunt him in the present. This happens with most of the suicidal patients – the past horrible experiences of the patients may continue to haunt him in his unconscious level. This may cause resistance on the part of the patient and treatment may suffer. In the clinical words, the transference to the clinician may have a major impact on the treatment, and counter – transference may also occur in subsequent time duration.(Gabbard & Allison, 2006) During the treatment period, practitioners have to face this situation and have to look into the patient’s past to bring out the root cause of his present situation and formulate this phase also. This process of integration of the past with the present is very painful thing for patients and in severe cases they can get emotionally disturbed, more depressive, anxious and can be aggressive, but nonetheless it is a temporary phase. I still remember she was nineteen years old and had gone into deep depression.When I asked about her past life, she entered into deeper state of depression and got completely silent and saddened and scared. I tried to relive her and promised not to ask about her past. She then slowly recovered herself, came back to normal and then after few days told me about her past. The treatment psychodynamic psychotherapy is all about the treatment of caring and love. In number of upheavals in our life, we need someone who can listen to us and care for us and here psychotherapist role starts. They listen to us and strive t o give us good hearing and relieve us from emotional pains.But, finally it is you only who is a healer and psychotherapist is only a guide who can take you on a self-guiding path. . Reference List Busch, F. N. & Milrod,B. L. 2008. Panic-Focused Psychodynamic Psychotherapy. Psychiatric Times. 25 (2). Corradi, R. B. (2006). Psychodynamic Psychotherapy: A Core Conceptual Model and Its Application. Journal of American Academy of Psychoanalysis, 34:93-116. Fink, B. 1997. A Clinical Introduction to Lacanian Psychoanalysis: Theory and Technique Harvard University Press. Gabard, G. O. & Allison, S. (2006). Psychodynamic Treatment.In Robert I. Simon, Robert E. Hales (Eds. ) The American Psychiatric Publishing Textbook of Suicide Assessment and Management: assessing the unpredictable. Arlington, VA: American Psychiatric Publishing, Inc. , 221-234. Gabbard, G. O. (2004) Long-Term Psychodynamic Psychotherapy: A Basic Text. Arlington, VA: American Psychiatric Publishing, Inc. McGrath, G. & Margi son, F. (2000) An Introduction to Psychodynamic Psychotherapy: BASIC PSYCHODYNAMIC CONCEPTS I. Retrieved on September 25, 2008 from W. W. W: http://www. geocities. com/nwidp/course/basic1. htmMcWilliams, N. (2004) Psychoanalytic Psychotherapy: A Practitioner's Guide. New York: Guilford Press. Shervin, H. , Bond, J. A. & Brakel, L. A. W. 1996. Conscious and Unconscious Processes: Psychodynamic, Cognitive, and Neurophysiological Convergences. New York: Guilford Press Wright, Elizabeth. 1998. Psychoanalytic Criticism: A Reappraisal. London & New York: Routledge. Yager,J. Mellman,L. & Rubin, E. 2005. The RRC Mandate for Residency Programs to Demonstrate Psychodynamic Psychotherapy Competency Among Residents: A Debate. Academic Psychiatry, 29:4, p. 339-349.

International Business Essay Example | Topics and Well Written Essays - 2250 words - 1

International Business - Essay Example This essay describes these benefits and hindrances, referring to further case studies as justification for the arguments provided. The advantages of diversification There are many risks of servicing only a singular market with one product or service that has been attributed to placing all of a firm’s proverbial eggs in a single basket (Thompson, Strickland and Gamble 2013). Virtually every product or service offered by a corporation has an established life cycle, moving from a growth phase to an eventual decline along the life cycle model in which sales and demand begin to decline. The life cycle of the product is determined by a number of factors, including consumer behaviour changes, innovative product releases by competition that outperforms, competitive pricing instances that drive price-sensitive buyers to rival firms, or even new market entrants that increase choice and lower switching costs for consumers to defect to a rival brand. Whatever the case driving life cycle, corporations must be keenly and proactively aware of the ability of their singular product or service in sustaining long-run profit growth. Because of the risks of a stagnating local market, businesses achieve advantages by diversifying the business into a new international market. The most significant advantage is that diversification allows the business to spread risks (Thompson et al. 2013). Risk occurs through a variety of drivers, both internally-related and externally-driven. For a business operating in a single market with a lone product, any changes to demand can impact revenue growth and even complicate many of the value chain elements that support business, including human resources, supply chain and procurement, as... As illustrated by the essay, there are many risks of servicing only a singular market with one product or service that has been attributed to placing all of a firm’s proverbial eggs in a single basket. Virtually every product or service offered by a corporation has an established life cycle, moving from a growth phase to an eventual decline along the life cycle model in which sales and demand begin to decline. The life cycle of the product is determined by a number of factors, including consumer behaviour changes, innovative product releases by competition that outperforms, competitive pricing instances that drive price-sensitive buyers to rival firms, or even new market entrants that increase choice and lower switching costs for consumers to defect to a rival brand. This paper makes a conclusion that there are actually more advantages than disadvantages in selecting a diversification strategy. Revenue increases, better cash flow position, and cost reduction in a variety of support divisions along the value chain are the most prominent of these advantages. Inclusive in advantages are better scope of control, more efficient and cost-acknowledging logistics opportunities, and even currency valuation in favour of the diversified corporation. The described disadvantages of unsubstantial cross-cultural knowledge of the foreign market, high control and power of buying markets, and disruptive innovation threats would tend to offset advantages when these situations occur in the new international market. Despite the disadvantages, the long-run benefits of diversification supersede the potential hindrances of seeking this strategy for growth.

Societies under Shogun RuleThe Ming & Qing Essay

Societies under Shogun RuleThe Ming & Qing - Essay Example This period, which started in 1368 until 1644, is marked by the decline of the decadent feudal system and the emergence of the embryonic capitalism in China. However, Japan’s shogun period was part of the early feudal system that would dominate Japan for more than 700 years. (Morgan 2003, 42) As both Ming/Qing and the Shogun societies were established as feudal societies above all, therefore, they share many characteristics. For instance, both had governments and rulers who enforced control over a highly fragmented society. The shogunate in Japan imposed guidelines for the creation and preservation of a stable national structure. This was achieved by implementing a highly hierarchical society, wherein the primary morality is based upon public rapport between master and subordinate. (Ratti & Westbrook 1991, 62) Such perspective determined the shape and functionality of the major social organizations of the shogunate societies. To illustrate: one of the rules/norms in the Japanese society then was that there was no more despicable crime than that of rebellion against a master. Because of this hierarchical social structure, the shogun was able to elevate the social structure which sealed the subjects of the nation into classes according to a vertical order of pragmatic impo rtance drawing heavily upon the military character and strength of the warriors to whom all other subjects were subordinated. The social pyramid follows this structure with the shogun and his class at the top. From 1615 onward, laws specifically determining the legal positions of and functions of the imperial court and its aristocratic families (Kuge sho-hatto), of the military class (Buke Sho-hatto), of religious orders (Jin-hatto), of the farmers (Goson-hatto), of commoners in Edo and, by analogy, in every town (Edo-machiju-sadame) were issued by the military government of the Tokugawa shoguns.

EasyJet Airline Company Essay Example | Topics and Well Written Essays - 2500 words

EasyJet Airline Company - Essay Example The timeline of EasyJet Airline reportedly started in March of 1995 as founder Sir Stelios Haji-Ioannou was noted to have offered flights at significantly low fares in Europe. Currently,it boasts of being â€Å"Europe's leading airline,operating on over 600 routes across 30 countries with our fleet of over 200 aircraft, employing over 8,000 people In its almost 18 years of organizational existence, EasyJet was noted to have been governed by a code of conduct to guide the behavior and decision-making processes of its personnel. The paper hereby presents a critical evaluation of the code of EasyJet Airline through a description of the code and by thoroughly assessing the code’s effectiveness in demonstrating the company’s values and conformity to business ethics. Critical Analysis of the Code The organization’s code was found under the title Governance on their official website: http://corporate.EasyJet.com/about-EasyJet/governance.aspx?sc_lang=en. Upon initial gl ance, the governance page contains information that indicates that the organization’s board of directors commissioned setting principles and ethical policies, of which, it was explicitly emphasized that the â€Å"key to these principles is working in an open and honest manner† (EasyJet Airline, Governance, n.d., par. 2). Likewise, the code was apparently patterned from the UK Corporate Governance Code which allegedly requires the following information to be disclosed:†¢ Terms of reference for the Finance Committee†¢Terms of reference for the Remuneration Committee... ance Committee   Terms of reference for the Remuneration Committee Terms of reference for the Audit Committee Terms of reference for the Nominations Committee   Terms and conditions of appointment / reappointment of the non- executive directors Matters reserved for the Board Non-Audit Services Policy (EasyJet Airline, Governance, n.d., par. 3). Finally, two (2) other documents were disclosed to be incorporated and accessible: (1) Corporate Governance Pages of the Annual Report; and (2) division of responsibilities of the Chairman and CEO (EasyJet Airline, Governance, n.d., par. 4). The Corporate Governance Pages of the 2012 Annual Report are found on pages 44 to 48 and contain the following major portions, to wit: principles statement, statement of compliance, leadership, directors’ conflicts of interest, board engagement with investors, board committees, relations with investors, internal control, risk management and internal audit (EasyJet, Corporate governance, 2012). T he Ethics Resource Center has recommended a code outline that should apparently contain the following format: I.  Ã‚  Ã‚   MEMORABLE TITLE   II.  Ã‚   LEADERSHIP LETTER   III.   TABLE OF CONTENTS   IV.   INTRODUCTION-PROLOGUE   V.  Ã‚   CORE VALUES OF ORGANIZATION   VI.   CODE PROVISIONS-SUBSTANTIVE MATTERS   VII.   INFORMATION AND RESOURCES (Ethics Resource Center, 2009, p. 1). From the course modules, it was explicitly defined that the contents of ethical codes are as follows: â€Å"standard of conduct, obeying the law, employees,consumers, shareholders, business partners, community involvement, public activities, environment, innovation, competition, business integrity, and compliance/monitoring† (University of Salford Manchester, What is the content of ethical codes?, 2012, p. 6; Crane and Matten, 2007).

Strategic Alliances Essay Example | Topics and Well Written Essays - 500 words - 1

Strategic Alliances - Essay Example In this respect, the DoT has sought to be involved as part of the multi-agency taskforce committee meant to monitor the project, as one of the important stakeholders to the project. This outsourcing caution resonates well with the outsourcing strategies provided under chapter 6, which requires that outsourcing can only make sense, where the outsourced services reduce the company’s risk exposure (Chapter 6, n.p.). Therefore, the caution issued by DoT to Department of Electronics and Information Technology (DIETY) of India, serves to meet this requirement as provided under the chapter. This is because, if the DIETY outsources manpower for the development of the NCCC, the private manpower will be aware of the possible security breaches that can be applied to bypass the security systems established by the project. This would be detrimental to the functionality and success of the project, considering that this project is meant to be applied for screening the entire web traffic on the internet within India, and then generate a security alert for the government recommending action, on the event that any cyber security threat is discovered (Aulakh, n.p.). According to the minister for information in India, the threat of Cyber security in India has been on the rise, with 24,216 Indian sites being defaced in 2013, compared to 17,306 sites that were defaced in 2011 (Aulakh, n.p.). In this respect, it is important that the government takes the necessary measure to curb the threat of cyber security attacks in the country, and that can only be achieved, if a foolproof system of scanning the threats is established. Nevertheless, the involvement of private manpower will be a higher risk to the project, than when the government enlists the services of its own manpower (Aulakh, n.p.). According to outsourcing strategies provided under chapter 6, outsourcing of

Enterprise Integration System Essay Example | Topics and Well Written Essays - 3000 words

Enterprise Integration System - Essay Example Forming a business application has proven to be tough and so is formulating an application to run an enterprise. The enterprise resource planning vendors have been successful in making humongous enterprise applications. Still the applications only account for a little percentage of the functions of a business that are required in an enterprise.The spread of these functions across applications renders the business more flexible in selection of packages for accounting, software that manage customer relationship and systems that process orders for the needs. The main aim of the selection done is to ensure that only the best is there for effective functioning. With the numerous requirements for the individual businesses, many applications will be highly recommended as compared to using a single enterprise application that performs all the functions.Vendors have found a way of adhering to this preference via offering focused applications to perform specific functions. However, the need th at arises of improving available software by adding their functions has affected other business applications. Users of the software do not put into consideration the limits of the functions of the systems whenever they perform activities with the businesses. These individuals perform the functions of a business regardless of the kinds of systems that the function entails. For example, when some one makes a call to alter his personal information and view if the last amount cashed went through.

Major Theorists Essay Example | Topics and Well Written Essays - 750 words

Major Theorists - Essay Example This pool of guilt and pain is where creativity, which is closely related to neurosis, comes from. Creativity is thus motivated by the need to express some of this guilt and pain in a more physical or outward arena as well as the means by which it is expressed. B.F. Skinner is one of the most well-known behaviorists, who believed that people were a product of the environment in which they were raised. Skinner suggested this occurred through operant conditioning as people were rewarded for approved behavior and punished for undesirable behavior. Rewards are thus seen as a means of strengthening behavior, even undesired behavior, while the failure to reward weakens these behaviors. In some situations, punishment can be interpreted as reward, such as when the ignored child receives attention through punishment for bad behavior. Creativity is thus seen as a mere product of the environment working on the individual who possesses latent creative talent. If the environment does not reward creativity, the child will be less likely to engage in it. It is important for the child to have an adult model that demonstrates creative behavior before the child can envision this as a likely activity for them. This is because they see the rewards for the cre ative adult as well as receive rewards from this adult as they engage in their own creative efforts. Humanism rejected the ideas of psychoanalytic theory of personality and creativity as a biological process and the ideas of behaviorism theory of personality and creativity as an environmental process and instead preferred to view creativity as a process in itself that provided individuals with the ability to discover their inner selves. In other words, creativity was an act of self-actualization. The main theorists of humanism are Abraham Maslow and Carl Rogers. Maslow defined self-actualization as the tendency to realize our own potential based on our

Corporate social responsibility and business Essay

Corporate social responsibility and business - Essay Example To answer the research question, there is consideration of the actual meaning of corporate social responsibility which relates to understanding the impact of business on the wider world, and considering the positive impact of the business. Corporate social responsibility is a bottom line determiner of the operation of the business. This is because a business that demonstrates enviable traits receives more customers since the business has a social obligation to its customer, implying that if the business demonstrates appropriate social responsibilities to the community which is also the customers, then they will feel much welcome to purchase from the business. In the study work of Boeger, Murray and Villiers (2008), there is evidence that taking a responsible attitude and going beyond minimum legal requirement without considering the size of the business are vital for the success of the business. The study further demonstrates that following straightforward principles that apply to an y business without considering the size is crucial for the welfare of the business. The research proposal takes into consideration the various forms of social responsibility and the bottom line impact on its success. ... The interview would entail recording of the information on questionnaires consisting of two sections. One section comprises information from the community where most are customers of the business, while the other section would be the information from the business. Most of the information in the reading is sourced as evidenced in the research work of Farquhar (2011). Literature Review Information and Data Analysis From the interview, it is evident that working with the local community comes with its benefits. From the information and previous study work of Heald (2005), many businesses find host in local customers since they are vital sources of sales. To be able to recruit employees, there is a need by the business to improve its reputation since a good relationship with the authorities and the community as a whole makes the operations of the business easy. According to Horrigan (2010), most local authorities award contracts to businesses which have a record of involvement in the com munity. Some of information from the interview explains that getting involved with the community would be in terms of supporting local charity or sponsoring a local event. This helps to prove the expertise of the business and demonstrate the face of the business. The information received explains that many businesses involve their employees while working with the community. This is in support to the research conducted by Jackson, Ones and Dilchert (2012), which explains that some businesses will let the employees choose and do voluntary activities for the community and avail pay off time for their efforts. Information received from the interviews indicates that corporate

Tiny toes ltd.How to start quality child care business Essay Example for Free

Tiny toes ltd.How to start quality child care business Essay Tiny toes ltd Introduction Importance of having accurate financial information in decision-making                   In the Tiny toes ltd, Sandra has no accuracy on the financial information. This makes the planning of the new projects of the new nursery and running of the business she has more difficult. Sandra needs to come up with accurate financial information on the finances of the business. It will help her have valuable data on the financial position of her company. It will also enable her determine its weaknesses and strength that intern assist her in evaluating the amount of capital needed to plan or start a new business (Way, 2014, n. p.). Apart from that, accurate financial information will help to maintain accuracy of taxes and ensure that additional duties imposed on the Tiny toes ltd are paid.                   Accurate financial information provides clear statement to investors in evaluating the business financial performance. By this communication will be available to the interested parties who want to contribute to the new projects of Sandra’s business. It will give them an easy way to determine accurate amount needed to finance the project completely. The investors and the contributors of the business rely on the accurate financial information for the safety and profitability of their investments (Way, 2014, n.p.). In the plan of starting a new business, she has to lay down the statements of finances for the budget needed like indentifying all income and expenses for the entire organization. She has to establish the time and period the budget will last and the capital required. By coming up with all this accurate financial information of Tiny toes ltd, she will able to access information on the past performance and the capital they have in hand. This will help to switch to another alternative incase a problem emerges in planning a new project.                   Consequently accurate financial information will help the banks and lenders to know if her business will be able to pay for the loan borrowed to avoid misfortunes in future (Way, 2014, n.p.). Apart from that, it will prevent the banks and lenders from stopping their support on her business through loans. Balance sheet is also part of financial information. Sandra needs to generate a balance sheet that will assess her business annually. The balance sheet will help determine the financial health of Tiny toes ltd. It will also help her have information on the productivity of the business and amount of capital that is retained. However, it will provide information on the flow of the business in terms of how fast or slow the assets are and how to convert them into capital. Tiny toes will be able to determine financial state they are in a specific time. With all the details of the financial information Sandra’s business will have a clear understanding o n how to come up with other new projects and how they will flourish.                   The benefits of Tiny toes having accurate cash flow in beginning a new nursery in GuildfordBusiness plans and cash flow also plays a big role on the progress of a business. This is what need to be initiated in Sandra’s a business to help the executives in the internal planning. The business plan will help in the application of loans and even borrow money from other lenders (Wasserman, 2010, p. 1) to support the projects. Lack of business plan in her business may lead to the downfall of Tiny toes ltd. By the business plan ahead, she will be able to identify the goals and objectives needed for the company to achieve its dream. However it will also help her determine the services needed to deliver in her project in Guildford. Provision of business plan for Tiny toes ltd will help to display the performance of her business and the goals ahead to start a new nursery and childcare centre.                   Another big role that the business plan plays is it reorganizes the business by making operational changes like reduction of expenses (Wasserman, 2010 n.p.). Nevertheless, it helps in the equity financing whereby banks and lenders are able to evaluate her business ability to take more loans if in need. As to cash flow, Sandra needs it to track and know the exchange of cash between Tiny toes and outside the business. The cash flow need to be done according to weekly and monthly basis to help monitor the flow of expenses and revenue. The cash flow is also important since it provides information on certain activities to notify Sandra so that she can know the impending danger in the business. In order to come up with accurate cash flow she should include aspects like cash receipts.                   The cash receipts include loans, private sources, fundraising and parents’ fees. However disbursement need to be included in the cash flow to provide information such as payroll expenses, supplies, maintenance, accounting and rent. All this details in the cash flow will help in calculating how much the company is making and if the profit incurred will help fund her new projects. Cash flow also helps in determining the changes in income and the balance sheet. The cash flow that Sandra needs, should be in different categories that is; operating activities, investigating, supplementary and financial activities (The globe mail, 2013, n.p). The investment cash flow will help determine changes on the long-term investment on the new nursery and childcare centre. It will also enable determine the rent needed in Guilford. Financial cash flow brings changes on stock needed and the payments needed for the interests of the shareholder.                   The operating cash on the other hand will enable Sandra view the records on the budget needed for starting a new business. All this shows how cash flow is very important in a business. It acts as a warning by identifying shortfalls in cash balances. It also helps in preparing budgets and helps sport payments problems by parents in the tiny toes business. If it is initiated in Tiny toes, Sandra will be able to know how her business generates profits and spends capital. It will also help her determine the technology improvements required in the new project of nursery and childcare centre. The business plan will help in good management her business. It will also help Sandra decide in her business whether to rent a new space in Guilford or not and initiates discussion of the objectives with the employees, partners and investors of her business. Problems and financial information that Sandra has to prepare and additional information needed to make a right decision                   The financial information of Sandra regarding Tiny toes and new projects of a new nursery and childcare centre is incomplete. In her information she only states the insurance, rent, rates, recruitment training, loans, wages, fixed prices and monthly charged price. She has left out the budget which is a very important thing in starting a new project. She has to come up with organizational and program budgets. Organizational budgets should indicate the income and expenses in Tiny toes ltd for a certain period of time (Child care center, 2009, p. 14). It should also show the anticipated expenses and income of the company since she wants to develop a childcare center.                   Sandra should give details of her budget in the way that it shows the expenses needed to start a new project of childcare center. It should also analyze expenses of each child according to time and the first class services offered to children from wealthy families. This will help in tracking the costs since everything is evaluated separately. In her information, she should state the time and period the budget will cover to determine amount required for it. Another thing that is not included in her financial statement is the report of the planner who will be in charge of monitoring the new business she wants to start. However, she has also skipped the aspect of licensing. Licensing may have many expenses that need high rate of funding (Mississippi, 2011, p. 11). It may include aspects such as safety codes and health care expenses because Medical examining is required for both the staff employed and children when dealing with a childcare center.                   Expenses for things like fire regulators also need to be included since they are the first priorities in her business to enhance safety. In her information she has only left out little details that seem to be not costly. When all of these little costs are put together they form a large expense that requires more help from the capital sector. By including all the details and coming up with an accurate financial information she will come up with her projects in a more efficient way. There will be no extra expense that will, destruct her planning. It will also help lenders and investors have clear information on the amount needed to fund the new projects. Problems experienced by Tiny toes in trying to generate internal finance in form of retained business                   Tiny toes have problems in generating internal finance due to the emerging competition which is increasing. The competitors that have nurseries provide the same services that Sandra’s company does. Their fees is less costly and due to that it poses a high risk to the progress of Tiny toes ltd. They are able to generate more capital than tiny toes and expand their services as compared to Sandra as a result of less cost. Due to this parents tend to take children to the competitors nursery than in Tiny toes ltd. Apart from that, Tiny toes will be forced to cut some costs that will drive away the staff and also students due lack of quality services.                   Lack of accurate financial information makes it difficult for her business to come up with a budget. However, a lot of capital is lost in things that are not necessary. Sandra’s business lacks plan and this is what destroys its capability to generate internal finance. Business plan is very important since investors are not able to invest in a business due to lack of clear information inform of cash-flow contained in it. This is the main things that that help in planning. Through business planning the Tiny toes will be able to understand how much profits the business will make and children needed in order to achieve more profits. This are the things that Sandra need to keep in mind in order to generate expand her business. Problems that Toes will encounter in the development of new projects                   Tiny toes will encounter problems in financial sector in the new project. This is due to high rent in Guildford and also adding more services only to capture the wealthy business may will bring loss to the business. In her projects she expands her business only to capture children who come from wealthy families. This shows they are not able to generate more internal profit. Tiny toes ltd does not expand its market share to capture children who come from at least less wealthy families. Relying on only one market share lowers the performance of the business especially in the financial aspect leading to low profit. Apart from that, tiny toes do not have accurate financial information. This shows that Sandra does not have information on the position of the business financially.                   In order for the Sandra’s business to prosper and generate more profit. She needs to expand her business by reflecting on both markets. She should focus on both wealthy and poor people. She needs also to keep in mind of the accurate financial information, business plan and cash flows in running the nursery and child care centers. She should also be aware of the competitors by coming up with more efficient plans that will make her secure her business and acquire a more large market share. Regarding the new projects she needs to take it slow by focusing on one project at a time. Focusing on both projects will lead to the downfall of her business since it will require a large amount of capital. Putting all this into consideration she will be able to plan other projects. Concentrating on one project at a time may save a company from bankruptcy. References Child care center (2009) financial planning and facilities development manual. Retrieved on 4th December 2014. http://www.buildingchildcare.net/uploads/pdfs/CCCManualFINAL07.pdf Mississippi (2011). How to start quality child care business. Retrieved on 4th December 2014. http://www.mdhs.state.ms.us/media/16085/pub_mp29.pdf The globe and mail.(2013). The importance of operating cash flow for small business. Retrieved on 4th December 2014. http://www.theglobeandmail.com/report-on-business/small-business/sb-money/cash-flow/the-importance-of-operating-cash-flow-for-small-businesses/article15134561/ Wasserman ,E.(2010). How to write the financial section of a business plan. Retrieved on 4th December 2014.http://www.inc.com/guides/business-plan-financial-section.html Way,J. (2014). What is the importance of financial statements. Retrieved on 4th December 2014.http://smallbusiness.chron.com/importance-companys-financial-statements-21332.html Source document

Paralleles of Cosi and Cosi Fan Tutte Essay Example for Free

Paralleles of Cosi and Cosi Fan Tutte Essay Cosi by Louis Nowra and Cosi Fan Tutte by Wolfgang Amadeus Mozart are set in completely different times but both themes of the play parallel each other. Cosi was set in 1970’s society whilst Cosi Fan Tutte was set in 1790. Cosi Fan Tutte is play about love and fidelity which parallels with what happens in Cosi. On the surface they are parallel because Lucy is unfaithful to Lewis when they are in a relationship together just as the women were in Cosi Fan Tutte. But it is much more than that. To really understand the parallels of the plays we must look at love and fidelity in depth. What really is love? What is demonstrated throughout both plays is that people have double standards towards love. In the Cosi we see free love and characters that believe in free love such as Lucy, Nick and Doug. Free love is the belief of having sexual relations according to choice, without being restricted by marriage or long-term relationships. They believe in freedom in doing whatever you want but this is the complete opposite of how they treat the mentally ill. They treat them with not dignity that makes them restricted to being behind walls of an asylum. Free love is about freedom yet they are confined to their ward like traditional marriage because people fear them. This is not the definition of love. Louis Nowra wants us to love each other and not stereotyping people by giving labels to people that we fear but instead showing respect and compassion. That is what true love is. In Cosi Fan Tutte we see two men test the love and fidelity of their girlfriends. This displays the double standards that men have on love. They believe that is alright to for them to test love. Mozart tells us that love needs to be equal that one side can’t unfaithful and that is alright. Mozart is trying to make us question what love is, like Louis Nowra did in Cosi. For there to be love, it must be equal and this is the key theme from bo th plays.

Concept of Influence in Leadership

Concept of Influence in Leadership Influence is defined as the force one person exerts on another person to induce a change in the targeted individual. Influencing can change a persons behavior, attitude, goals, opinions, needs and values. Influence is a necessary part of leadership. It is concerned with how the leader affects followers. Influence is a necessary ingredient of leadership, without influence, leadership is non-existent. The influence dimension of leadership requires the leader to have an impact on the lives of those being led. To make a change in other people comes with an enormous amount of social and ethical burden. Various types of influence processes and the factors affecting them Leaders use variety of influence process to alter the behavior of people. The influence process changes according to the demands of the situation. Influence processes refer to the five ways leaders shape organizational variables including people and resources. The five influence processes are direct decisions, allocation of resources, reward system, selection and promotion of other leaders and role modeling. Each will be discussed in detail in the following paragraphs: Direct decisions:Â  Direct decisions provide the leaders the ability to influence the choices of their followers. This ability to influence comes with the control leaders have in formulating mission and vision aspects of an organization. The most important aspect giving power to leader in the organizational structure is their ability to influence the variables affecting their followers through direct decisions. Leaders ability to make decisions influencing organizational structure give them the power to dictate decision making in the organization and set overall direction of the organization. Allocation of resources: Leaders make use of their power over various organizational resources such as human, money and technological resources to significantly impact the way work is carried out in an organization. For example if a department is in serious need of funds a leader has the power to grant additional money to the department in addition to the portion allocated to the department in the annual budget. Reward system: Leader can make use of their power to reward to influence the actions and behaviors of their employees. A leader can offer monetary incentive to followers in case the followers conform to certain behavioral standards and achieve goals necessary for meeting the overall organization objective. Selection and promotion of other leaders: Leaders can also influence their followers by making use of their power to promote the leaders who meet individual as well as organizational goals and objectives. Role modeling: Finally one of the most important ways leaders can mould the behavior and opinion of their followers is by acting as a role model. Followers always follow the path leader is taking. If a leader possesses high moral and ethical standard there are greater chances that their followers will also have high ethical and moral standards. Analysis of the influence process used by Andrea Jung, Indra Nooyi, and Brenda Barnes Influence processes used by Andrea Jung Direct decisions: The CEO of Avon Corporation, Andrea Jung has taken numerous successful decisions that have changed the image of Avon in the beauty and cosmetics industry. She emphasized the importance of sales force saying that they were at the heart of Avon and that Avons future success depends on their efforts. She also strongly emphasized the importance of internet sales. Andrea Jung also worked to change Avons traditional focus from the 30-55 year old women to the younger 16-24 year old women citing the reason that this segment of around 17 million women has a purchasing power of over $100. She also eliminated seven layers of management and got rid of 25% of the senior staff. Allocation of resources: Andrea Jung strongly favors allocation of huge budget for the Research and development function of her organization, believing that it has potential to give off higher benefits in return. Andrea Jungs decision to invest heavily in RD paid off when the companys sales increased by 45% in 2004. Reward system: Sales representatives making huge profits for the company are rewarded with paid holiday trips to eye-catching tourist attractions like Tenerife, Cyprus and Madeira. Selection and promotion of other leaders: When Andrea Jung took over Avon Corporation there were no women past middle management. Today 85% of Avons top management team in U.S. being composed of females ([emailprotected], 2005). Role modeling: Andrea Jung with her determination, courage and distinctive managerial style is a wonderful role model for coming generations of female leaders. Influence processes used by Indra Nooyi Direct decisions: At PepsiCo. Indra Nooyi has been credited with several key decisions that have positively enhanced the performance the company. Her decisions including the divestiture of fast food restaurants (Taco Bell, Pizza Hut and KFC), the acquisition of Tropicana and the merger with Quaker Oats provide remarkable examples to the sharp talents Indra Nooyi has in turning ideas and plans into realities. Allocation of resources: Indra Nooyi redirected about one third of PepsiCos marketing budget this year towards social media campaigns and digital promotions including her ReFresh Project campaign. Indra Nooyi takes community building seriously and has undertaken a $20M grant program to help community building causes. Reward system: Apart from monetary rewards, Indra Nooyi strongly believes in the importance of non-monetary rewards as well. She sends the spouses and parents of her companys top performers personal hand written thank-you letters (Keller Price, 2011). Selection and promotion of other leaders: Indra Nooyi stresses the importance of developing future leaders so that when the top level leaders leave their jobs their absence is not felt (Keller Price, 2011). Role modeling: Indra Nooyi is a role model for millions of young girls back home in India. It is a signal to them that with extreme dedication and hard work they too can scale apparently unreachable heights. Influence processes used by Brenda Barnes Direct decisions: The CEO of Sara Lee has undertaken number of direct decisions during her tenure at the company. Her first major decision was to sell its non-core businesses to focus on the core business. She also decided to increase Sara Lees presence in the international market, because of this decision in 2008, 75% of the companys operating income came from international (non-U.S.) market. Allocation of resources: When Brenda Barnes first assumed the role of CEO of Sara Lee she took the decision to allocate $250m towards RD and marketing in addition to the allocated amount in budget, she undertook the decision to boost the annual sales at least by 2% to 4%. Reward system: At Sara Lee rewards are tied to employees performance on job. Employees are encouraged to buy companys stock and naturally it gives provides them an incentive to perform exceedingly well on their jobs since it will translate into profits for them (Griffin Moorhead, 2011). Selection and promotion of other leaders: Brenda Barnes promoted leaders to higher echelons of the organization depending on their skills and performance level on job (Griffin Moorhead, 2011). Role modeling: Brenda Barnes is a great role model for women looking for finding a balance between career and home. She took a career break and left her job as the CEO of Sara Lee for looking after her children (Bulik, 2004). Conclusion Influence is the force a person exerts on another person to change their opinion or alter their behaviors. Leaders frequently use a combination of influence strategies. It is a well-known fact that people who use a wider variety of tactics are typically perceived as having greater power and influence. Leaders have to determine the method that is most likely to influence others by considering the individuals, groups, and situations involved.

Film: 2001 :: Papers

Film: 2001 2001 is a masterpiece of cinema that still influences film makers nearly thirty years after it was made -- but what does it actually mean? Therein lies the enigma. Of course, 2001 is open to many interpretations and probably even Kubrick couldn't provide the "correct" one. The film is very different from the book; Kubrick reduced the original script to its bare essentials making the actors part of the narrative , but not telling the narrative through the script. making it a lesser part of the hole experience. Where there is speaking it is almost always symbolic The first words spoken signal the decay of human language to empty phrases: "Here you are, sir. Main level, please." The opening of 2001 is the Dawn of Man sequence which dovetails neatly with end of Dr. Strangelove: "We'll meet again, some sunny day " First image in the film is of a rising Sun Obviously, Kubrick pondered deeply the astonishing reality, that idea that man was smart enough to blow up the earth, but not smart enough to stop that from happening (kubric)(man doesn't want to nail himself, but he does). How could such a phenomenon occur? With such strong symbolic events and imagery in the opening seen it is hard to see them all as individual events, kubric uses these to tell the narrative of the story. The Sun is not just light, but heat (a desert). Making the Sun not necessarily good, the Sun is usually seen as positive in relation to dark, but not in a desert. This makes the sun a negative, with the use of water as a positive. The leopard killing the zebra Is a key element to the opening scene representing the behaviour of man the Zebra is a coexistence of black and white? Good an bad together just like man, making the leopard the destruction of man kind maybe symbolising the bomb. To echo the directors words ,QUOTE "you're free to speculate as you wish about the philosophical and allegorical meaning of the film" but

Animal Experimentation :: Ethical Issues, Persuasive

Animal experimentation is a controversial topic and may what to stop the use of animal in experiments. Experimenting with animals helps save human life but innocents animal pay the consequences. The "pro" and "con" positions on animal experimentation have proven suitable for stating extreme opinions, but unhelpful at exploring the broader ethical questions of animal based research (Wolff, 2009). Human and animals are very similar, for scientific is easy to use animals to find cures for humans decease. It difficult to scarified innocent animals but those animals help human to continue living. While there are multiple accusations of animal cruelty, few people actually witness the inner workings of a research lab and multiple regulations prevent animal cruelty, but animal research still continues to cause pain (Wolff, 2009). The use of animals in experiment is fundamental for human descendent. Many Humans are absolutely positive that scientific should experiment with animals to save humans life. Because there are so many similarities between human and animal physiology, experimentation has allowed scientists to discover new treatments for diseases and guarantee the safety of new drugs (Foundation for Biomedical Research, 2009). Experiment with animals is necessary because without the animals scientific are not able to find cure disease. Animal investigation has offered the source for several medical innovations, including vaccines, organ transplants, and pacemakers (Foundation for Biomedical Research, 2009). In this world are many diseases that did not have a cure but there are other diseases that have it and those cures were found with the help of the animals experimentations. â€Å"In 2006, infant mortality in the USA—a key indicator of the nation's health—was measured at fewer than seven deaths per 1,000 live births compared to 47 deaths per 1,000 live bi rths in 1940, much of this progress came from knowledge gained through animal research, many diseases that once killed millions of people every year are now either preventable, treatable or have been eradicated altogether, immunizations against polio, diphtheria, mumps, rubella and hepatitis have saved countless lives, and without animal research, these vaccines would not exist (Foundation for Biomedical Research, 2009).† Also, experiments on animals are necessary in order to safeguard and improve human health and well-being, and vaccines for diseases such as polio and malaria could not have been developed without animal testing, Cohen explains (Cohen, 2005). Animals are crucial for finding cure for those Children and adults that need an opportunity to continue living. â€Å"This true story, close to us in time and place, is widely known.

nationalism :: essays research papers

Founded in 1965 by Ron Karenga, US emerged in Southern California shortly after Watts exploded into a riot and Malcolm X was assassinated. As nationalist sentiment grew among African American organizations because of the impact of Malcolm X, US established itself as a cultural nationalist group. It called for racial unity and for black people to free themselves from white oppression by embracing a "recovered" African culture. For US, that meant Kawaida, a quasi-religious system of beliefs and rituals advocating black pride, unity, culture and self-defense. Upon becoming a member or "advocate" of US, a person was given a Swahili name, urged to wear African clothing and immersed in the group's complex doctrine, practices and organizational hierarchy. Karenga, a budding scholar of African studies who was fluent in Swahili, constructed Kawaida, says Brown, by adapting rituals and beliefs -- primarily from the Zulus of South Africa -- to the organization's own rituals, beliefs and holidays. Kwanzaa, a holiday created by US in 1966, is now observed by millions of African Americans. Brown's portrait is historically sharp and honest. He includes a discussion of the damaging effects of sexism and of Karenga's cult of personality. Brown's research is also sensitive to misconceptions that have plagued US' past, such as the name standing for "United Slaves." The name, Brown says, simply "stands for Black People: the pronoun 'US' as opposed to 'them,' the white oppressors." Brown's study of US emerges as a keen observation of how a relatively small group became a central force in a mass movement through its ideological influence. "The group's approach to organizing," Brown writes, "which resisted mass recruitment into its ranks . . . saw no need for a large membership. Their goal was to ideologically influence other organizations with its united- front approach, and thus direct the course of the coming 'cultural revolution. ' " Brown illustrates this best in the section "The New Ark Laboratory," in which he cites US' alliance with writer-activist Amiri Baraka and several organizations in Newark, N.J., which not only helped elect the city's first black mayor but also turned Newark into a stronghold for the Kawaida doctrine when US fell into decline in the early '70s. Brown is equally attentive to the stumbling blocks faced by US. For instance, the section "Operational Unity and the US-Panther Conflict" gives a dynamic history of the escalating tensions between US and the Black Panther Party, as well as Karenga's political missteps that jeopardized US' anti- establishment credibility. nationalism :: essays research papers Founded in 1965 by Ron Karenga, US emerged in Southern California shortly after Watts exploded into a riot and Malcolm X was assassinated. As nationalist sentiment grew among African American organizations because of the impact of Malcolm X, US established itself as a cultural nationalist group. It called for racial unity and for black people to free themselves from white oppression by embracing a "recovered" African culture. For US, that meant Kawaida, a quasi-religious system of beliefs and rituals advocating black pride, unity, culture and self-defense. Upon becoming a member or "advocate" of US, a person was given a Swahili name, urged to wear African clothing and immersed in the group's complex doctrine, practices and organizational hierarchy. Karenga, a budding scholar of African studies who was fluent in Swahili, constructed Kawaida, says Brown, by adapting rituals and beliefs -- primarily from the Zulus of South Africa -- to the organization's own rituals, beliefs and holidays. Kwanzaa, a holiday created by US in 1966, is now observed by millions of African Americans. Brown's portrait is historically sharp and honest. He includes a discussion of the damaging effects of sexism and of Karenga's cult of personality. Brown's research is also sensitive to misconceptions that have plagued US' past, such as the name standing for "United Slaves." The name, Brown says, simply "stands for Black People: the pronoun 'US' as opposed to 'them,' the white oppressors." Brown's study of US emerges as a keen observation of how a relatively small group became a central force in a mass movement through its ideological influence. "The group's approach to organizing," Brown writes, "which resisted mass recruitment into its ranks . . . saw no need for a large membership. Their goal was to ideologically influence other organizations with its united- front approach, and thus direct the course of the coming 'cultural revolution. ' " Brown illustrates this best in the section "The New Ark Laboratory," in which he cites US' alliance with writer-activist Amiri Baraka and several organizations in Newark, N.J., which not only helped elect the city's first black mayor but also turned Newark into a stronghold for the Kawaida doctrine when US fell into decline in the early '70s. Brown is equally attentive to the stumbling blocks faced by US. For instance, the section "Operational Unity and the US-Panther Conflict" gives a dynamic history of the escalating tensions between US and the Black Panther Party, as well as Karenga's political missteps that jeopardized US' anti- establishment credibility.

Describing Death

Giovanni Martinelli’s early career as a Florentine painter is relatively unknown. It is documented that he moved to Florence in 1634. Prior to relocating, Martinelli produced Memento Mori (Death Comes to the Dinner Table). This piece has a comparable style to the Florentine painters Filippo Tarchiani and Anastagio Fontebuoni. Memento Mori is displayed in a clear narrative style. Martinelli created a colorful and dramatic scene that was underscored by the hasty arrival of death. Some translations state that Memento Mori means â€Å"Remember, you shall die. Here, Martinelli portrays a group of five individuals of varying ages. They appear to be from a wealthy stock. They wear expensive clothing and are seen at a dinner table partaking in a party of sorts. They are gallivanting around a table that is filled with an abundant collection of exotic fruits, pies, and pastries. Along with the food, Martinelli painted various wine glasses that appear to be hand-crafted and of the highe st fashion. Giovanni used bright, shiny silken wear with colors ranging from yellow, to peach, to blue. The colors complement each figure, adding to their personality and desire for greed. These wealthy-looking people appear—almost instantaneously—to be in a rigid state of duress. On the far right side of the painting, we see, in dark shades, the resemblance of a skeleton holding up an hourglass, as if to say, â€Å"cheers. † This one-time jovial band of feasters is seen reacting to the new character intruding on their high-standing party. The faces offer gestures of utter shock and dismay. All the focus shifts from their colorful mealtime festivities to this arch nemesis, and back again. The skeletal metaphor of death leans into one man’s shoulder. This man closest to death is in the act of clutching his heart. His eyes dilate toward the figure of death and his mouth, just slightly ajar, appears to be ready to cave open in utter horror. He, however, is too dumbfounded by this apparent figure of death’s close proximity to do anything but freeze in horror. ANALYSIS OF MEMENTO MORI There are two men at the rear end of the table, furthest from death. They are also horrified. One man is seen posturing with arms wide-open, even though his is furthest from death. He is attempting to back away; yet, at the same time, he is powerless and unable to leave the empty gaze of death’s vision; for running will do no good to the mortal man, regardless if he led a life of sin-hood or martyrdom. Morals were almost epidemic in Martinelli’s fresco works. Memento Mori is another classic story-telling scene with moralistic intent. The era of this 17th century creation occurred at a time when plague was a hearty, healthy killer. The deathful onslaught of plague, in all its pestilence, came to towns without warning. It attacked the poor and wealthy alike, caring little for a person’s status in regards to wealth, politics, or religion. To create the moral fabric surrounding the powers of death through plague, Martinelli displayed deep imagery between the youthful party-goers who were enjoying their food and intoxicating drinks. He contrasts these free-willing, wealthy individuals with the ghastly—even chilling—reality of death. He does so by casting death’s chin over one man’s shoulder. This imagery adds power and personality to the fresco. Martinelli displays the image of death coming to this gathering alone and without emotion or guile. The lone figure of death is shown to uproot the lives of this group of healthy, stress-free people—startling the living daylights out of everyone, young and old. MODERN-DAY IRONY Today, some four-hundred years after Giovanni Martinelli finished his last brush stroke to canvas, we remain as close to the fear of death’s randomness as the 17th century plague-ridden societies were. For 300 years plague hung over the lives of Europeans like an omnipresent cloud,† said Mormando, who is an Italian studies professor at Boston College. This statement, today, holds tremendous weight to our real-world current lifestyle. Today, terrorism, war, suicide bombing, and the like are the molds that cast this same skeletal image at our kitchen table. Terrorism is a specter that comes at us in many forms: anthrax, subway bombs, and suicide hijacker leading to the massive death of September 11, 2001. Just as smallpox left past centuries in a state of despair and real fear, we, today, see how fear forces us to change our reality. This changing reality holds an ominous likeness to the changes that the banqueters in Momento Mori faced. Martinelli’s plague painting is characteristic 17th century Italian. The horrific tendency of the subject matter never destroyed the utter beauty—even charismatic charm—of the oils that he chose to mix in his representation of terror and deathly destruction. For in his era of epidemics and massive death, hardly a decade passed without plague haunting a town and bringing it to its knees. When this painting was created, there wasn’t anything small about death. It came in massive, near apocalyptic proportions, decimating generations in the blink of an eye. So, the timing of Martinelli’s piece was right in line with the mood of the generation. This connection further enlivened his work back then. Since we, in the dawn of the 21st century, can relate to massive death and fear through terrorism, war, and civil upheaval around the globe, it also adds empowering imagery to his age-old mastery. The imagery of Memento Mori is further encapsulated by the grave images on these wealthy party-goers’ faces. The hand-crafted beautiful likeness on the faces of those facing death offers deep-rooted thinking that says: As some of the Italians survived a plague-ridden epidemic, they continued to look-over-their-shoulders awaiting the next surprise visit by the specter of death. It’s rather haunting how this parallels with what terrorism instills upon societies around the planet today. Plague paintings like Martinelli’s, many times, depicted a person pinching their nose to refrain from smelling the horror of death in the air. Martinelli, however, appeared to glaze over his moral of death and the fear of death by not rendering any people in his paintings to be abhorred by the scent of death. This may further enhance the true lifestyle of the wealthy in the 17th century: they had more than they could fathom. So, was Martinelli a closet optimist? Or was his subliminal imagery set up to leave us with this question: How can the rich continue to ignore the fate of death, and why don’t they seem concerned with finding ways to better humanity instead of bettering their personal property and lavishness? In closing, we must also consider the element of hope and healing, and why it is not apparent in Memento Mori. We must consider the ‘miasma theory’ and its transmission by way of corrupt air. It’s the complete randomness of plague that Martinelli depicts so well that adds such force to this painting. As we continue to live in corrupt societies, it seems that the fear of death will continue to hang over our shoulders. Terrorism is likened to the countless plagues that washed over communities those many centuries ago. As death and terror come and go, the art of the masters remain untouched.

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. (No 72. 7. 0135), to a generous contribution of the Fondation pour la Recherche Mf? dicale Franpise and to a participation of the CEA (Saclay, France) in the purchase of radioactive compounds. The work has been performed as a partial fulfillment of a thesis (Doctorat Bs-Sciences) submitted by L. D. -K. Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser REFERENCES 1. Robison, G. A. , Butcher, R. W. & Sutherland, E. W. (1968) Ann. Rev. Biochem. 37, 149-174. 2.Walsh, D. A. , Perkins, J. P. & Krebs, E. G. (1968) J. Biol. Chem. 243, 376 3-3765. 3. Kuo, J. F. & Greengard, P. (1969) Proc. Nut. Acad. Xci. U . S. A. 64, 1349-1355. 4. Reimann, E. M. , Brostrom, C. O. , Corbin, J. D. , King, C. A. & Krebs, E. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. 5. Tao, M, Salas, M. L. & Lipmann, F. (1970) Proc. Nut. Acad. Sci. U . S. A. 67, 408-414. 6. Gill, G. N. & Garren, L. D. (1970)Biochem. Biophys. Res. Commun. 39, 335-343. 7. Craig, J. W. , Rall, T. W. & Larner, J. (1969) Biochim. Biophys. Acta, 177, 213-219. 8. Stuil, J. Mayer, S. E. (1971) J. Biol. Chem. 246, 5716-5723. 9. Schaeffer, L. D. , Chenoweth, M. & Dunn, A. (1969) Biochim. Biophys. Acta, 192, 292-303. 10. Miller, T. B. , Exton, J. H. & Park, C. R. (1971) J. Biol. Chem. 246, 3672-3678. 11. Harbon, S. & Clauser, H. (1971) Biochem. Biophys. Res. Commun. 44, 1496-1503. 12. Corbin, J. D. , Soderling, T. R. & Park, C. R. (1973) J. Biol. Chem. 248. 1813-1821. 185 13. Soderling, T. R. , Corbin, J. D. & Park, C. R. (1973) J. Biol. Chem. 248, 1822-1829. 14. Gill, G. N. & Garren, L. D. (1969) Proc. Nut. A d . Sci. U. 8. A. 63, 512-519. 5. Walton, G. M. & Garren, L. D. (1970) Biochemistry, 9, 4223-4229. 16. Gilman, A. G. (1970) Proc. Nut. Acad. Sci. U. 8. A. 67, 305-3 12. 17. Do Khac, L. , Harbon, S. & Clauser, H. (1973) Ninth Int. Congr. Biochem. p. 354. 18. Lowry, 0. H. , Rosebrough, N. J. , Farr, A. L. & Randall, R. J. (1954) J. Biol. Chem. 193, 265-275. 19. Wilchek, M. , Salomon, Y. , Lowe, M. & Selinzer, Z. (1971)Biochem. Biophys. Res. Commun. 45,1177-1184. 20. Chambaut. A. M. , Lerav, F. & Hanoune, J. (1971)PEBS . . â€Å". Lett. 15,'328-334. Walsh, D. A. , Ashby, C. D. , Gonzalez, C. , Calkines, D. 21. Fisher. E. H. & Krebs. E. G. (1971)J. Biol. Chem. 246, i977-1985. 22. Ashby, C. D. & Walsh, D. A. (1973) J. Biol. Chem. 248, 1255-1261. 23. Haddox. M. K. , Newton, N. E. , Hartler, D. K. & Goldberg, N. D. (1972) Biochem. Biophys. Res. Commun. 47,-653-661. 24. Klotz, I. M. (1953)in The Proteins (Neurath, H. & Bailey, K. , eds) p. 772, Ac ademic Press, New York. 25. Cleland, W. W. (1967) Advan. Enzymol. 29, 1. , L. Do Khac, S. Harbon and H. J. Clauser, Institut de Biochimie, Universit6 de Paris-Sud, BLtiment 432, F-91405 Orsay, France Eur. J. Biochem. 40 (1973)