Recommended procedures for improvement Essay

As systems turn out to be more programmed and mechanized, those in charge of information system have a tendency to consider that more power will construct their processes better organized. In pulling manufacturing and design en route for computerization, the inclination is to provide as much power as probable to the mechanism and leave the lingering job tasks at hand. This automation viewpoint reduces the information and perceptive capacities of workers and pushes them to dislike the technology. Premises in the field of information system management are significant whether one’s orientation is in the direction of research or practice (Tapscott, 3). The responsibility of an organization is to help out in withdrawing the complication of the world and explaining and predicting proceedings. Given the information that the purpose of information system management management is to lessen the convolution of the empirical world by means of technology can benefit from sound improvement. While the complexity of the information system plan will vary based on the size of the district, it has to include a mission statement and rational, quantifiable goals and objectives that can be attained, in most cases, with obtainable resources. The Alachua’s budget also has to reflect the financial obligation to major technology initiatives included in the plan. Additionally, the planning process must include proceedings that allow decision makers to gauge the benefits of investments in information system and abandon or alter failed strategies for more triumphant ones. Methods of making a Web site secure from hackers – It is not that software has turned out to be less protected, and it’s not almost certainly because of potential attackers having increased in number. The reason that hackers are more common is likely because many people do not know how to protect their Web sites. To make a website free from hackers, simply: (1) know your enemy, (2) create ‘strong’ passwords, (3) use good software, (4) update recurrently, (5) use a hardware server-side firewall and (6) facilitate WPA or WEP encryption on wireless network connections. Digital Signature – Digital signatures are just like written signatures – these are used to provide verification of the related input, typically called a ‘message’ – (may be anything) a message sent in a more complicated cryptographic protocol or from an electronic mail to a contract. In cryptography, a digital signature or is a kind of asymmetric cryptography used to reproduce the security properties of a signature in digital – than in a written form. Digital signatures usually give two algorithms – one for signing that involves a user’s secret (private key), and one for validating signatures that involves user’s public key and the output of the signature method is known as the digital signature. â€Å"Terms and Conditions† or â€Å"Conditions of Use† -Websites and its related services are administered by the Terms and Conditions of Use, and the constant access to websites and its services is put through the user’s agreement to be bound by the Terms and Conditions of Use, as they may alter occasionally. Terms and Conditions of Use is a provision placed on a website notifying users regarding how the website deals with a user’s rights and responsibilities. Some websites characterize their Terms and Conditions of Use allowing users to routinely assess its services offered. If a user does not agree to Terms and Conditions of Use, a user may not use the website or its services. Plans of Alachua County in information system must state who is responsible for implementing and updating the technology plan. The Alachua County District’s technology plan must includes information on individual needs, equitable resource allocation, cost-effective acquisitions, technology funding, infrastructure and networking issues, technology-related professional development, support needs, and information management and delivery. On the other hand, the district’s does not make use of this best practice for the reason that the plan is not complete, is not based on a valid needs assessment, and is not evaluated and updated in a scheduled manner. The constituency should guarantee that the goals listed in the plan are detailed, quantifiable, manageable, rational, and time-bounded. The county also should ensure that the plan is monitored and updated based on valid data related to the district’s technology needs, skills, usage, and fluency levels among district employees. Strategies in the county’s strategic plan relate to technology and are considered the goals for the technology plan. In the district technology plan, the objectives are ambiguous (e. g. : guarantee, renovate, generate, etc.). Few of the objectives are assessable and are not based on detailed standards, making it complicated to document whether the objective was acquired. Additionally, it is imperative to note down that all decision-making in information system management is derived from some embedded premise. For the reason that the consequences by means of vague assumption information system management are better for all – assumptions that give understanding may perhaps have even greater importance to all organizations (Tenkasi, 27). If one relies barely on the capability to forecast events devoid of understanding them, there is a greater prospective for making underprivileged, or at least unproductive decisions. The recommendations given are good examples of research that has hardened models of managing information systems in local areas and even in worldwide perspective. Yet there is still a need for more investigations to be able to accomplish a more constructive course of action in business information technology management. Information technology systems and management necessitate of a solid abstract establishment to direct both practice and research. Even though many of the recommendations being proposed by scholars and scientists in this field may possibly be compelling prescriptions for those who are on the lookout for something further than unsystematic information management, unless information management turns out to be strongly stranded in theories of organization. Works Cited: Tapscott, D. (1998) Growing Up Digital:The Rise of the Net Generation. McGraw Hill. Tenkasi, RV (1995) Technology transfer as collaborative learning. NY: Free Press Wesier, P. J. (2003). The Internet, Innovation, and Intellectual Property Policy. 103 Colum. L. Rev 534.

Jefferson Essay Essay

Thomas Jefferson played a very important role in the history of the United States. Jefferson is most famously known for writing the Declaration of Independence. Thomas Jefferson talked many times about African-Americans in America. Where they equal to white people? How were whites and blacks different? What about slavery? Thomas Jefferson had an opinion on all of these subjects, but much of what Thomas Jefferson said was later contradicted with his own words. What did Thomas Jefferson mean when he said that all men were equal? Well, many people would agree with what it means today in modern times. Today the statement, all men are created equal means; all men and women whether black, white, tall, short, fat, or skinny were all created equal. That is not necessarily what it meant in the 1700s. Blacks were enslaved and worked hard while wealthy white men did nothing of the sort. Justice Thurgood Marshall said, â€Å"The blacks were so far inferior, that they had no rights which the white man was bound to respect†¦ and that the Negro might justly and lawfully be reduced to slavery for his benefit. This excerpt shows that in that time blacks were not treated the same as whites and clearly did not have the same rights. Conor Cruise O’Brien’s book confirms this belief as he takes a quote from Thomas Jefferson himself: â€Å"It is accepted that the words ‘all men are created equal’ do not, in their literal meaning, apply to women, and were not intended by the Founding Fathers to a pply to slaves. † This shows that when Thomas Jefferson talks about all men being created equally he means to say white men. The meaning of the word expatriation is as follows; to leave one’s native country to live elsewhere (Merriam-Webster). Jefferson wanted to send the blacks that were descendent of the original slaves back to Africa. The deal here was Africa was no longer their native country. This act was known as expatriation. The southern courts believed that slavery violated the natural rights of blacks and although slavery was allowed and legal it was immoral and unjust. Thomas Jefferson made it known that he supported the abolishment of slave trading. He said the following to Congress; â€Å"withdraw the citizens of the United States from all further participation in those violations of human rights which have been so long continued on the unoffending inhabitants of Africa. † By this Jefferson means that the people of the United States should stop the enslavement of blacks so that they no longer violate the human rights and stop offending the people of Africa. In this way Jefferson’s reasons for promoting expatriation and understanding for the natural rights of blacks were consistent. He wanted to send the blacks back because he wanted to stop offending the people of Africa. Jefferson believed that blacks and whites were very different. He said that he believed that blacks were originally their own race and became distinct by time and certain circumstances. According to Jefferson blacks came from their own species but were from the same general genus that is the human race. He said that blacks were inferior to whites in mind and body. This can be said to mean that whites were possibly better looking people or that they were more fit. It could have been said to mean that whites were smarter than blacks because they had an education that the blacks did not have. Jefferson did not believe blacks were men. â€Å"Men,† was said to be referred to as only white men. Samuel Eliot Morison says in his book; â€Å"In his views (Jefferson) blacks were not men. † The possession of individual rights was mainly determined by one’s race or skin color. Blacks had no rights early on in the 1700s but began to become free in the early 1800s. A wealthy white male had all rights; he was allowed to do what he wanted so long as it was just. A black man was free and eventually gained simple rights due to the belief that the United States was violating human rights. I do not believe that expatriation could have been a helpful solution to the problem of slavery in America. I believe this because if the slaves would have gone to Africa they would be gone, but there is no saying that the lazy southern farmers would not have brought in slaves from other places. They would have gotten so used to not having to work that they would not be able to start working again. I think that they would possibly keep indentured servants as permanent slaves or enslave poor people. I’m sure that expatriation would not solve the problem of slavery, at least not immediately. As I stated in the beginning, Jefferson’s view changed greatly throughout the course of his lifetime. Later in his life he began to appreciate black people more because of all their hard work. Also, he realized that blacks were the same as whites in that they were to be given human rights and that the people of the United States had been violating their rights from the very beginning. Jefferson also saw that blacks were men, and that really God created all men equal, black and white. Thomas Jefferson certainly lived an intriguing life. He lived during a time where slavery was legal. He lived during a time when slavery began to die out. Jefferson was part of the reason why blacks are free today. Although his beliefs shifted throughout the duration of his life he ended on the good side, the side that was just. He began to believe that all men are created equal with a literal meaning.

Entifying Information Assets, Threats, and Vulnerabilities in Top Assignment

Entifying Information Assets, Threats, and Vulnerabilities in Top Information Security Breaches of the Decade - Assignment Example If is not resolved, it can lead to very serious information security. The electronic or the physical security systems are not the organization’s weakest security link; human are the one with the weakest links. It is always assumed that the insider threats originate from rogue workers or planted ‘moles’, IT administrators and managers who are privileged to the access of sensitive information, controls and resources poses the greatest risk. They can start and stop a system, make changes that are critical such as giving the rights to access and untraceably deleting security logs. This threat is devoted to describing the mechanisms used for compromising organizational intellectual property from within (Gupta, et al. 2012). A lot of security incidents are caused by insider misuse i.e. accidental or malicious. A lot of misuse occurs within the boundaries of trust necessary to perform duties. Preventing the misuse is difficult as the only way to stay secure is to grant a ccess rights only to those with business need and to keep an eye on their activities. The problem is that the majority of organizations have very limited capabilities to trace specific IT events to specific users, with any certainty. A small fraction of IT teams are aware of what is going on in their infrastructure in a particular time and some organizations look manually through files so as to get answers. Unauthorized access by insiders; by accessing the organization’s secured areas physically, or materials that have data which is sensitive make it very easy for a crime to be committed by malicious insiders. The physical security measures of an organization are as important as the technical security controls. This threat is emerging as a great risk to corporate data. The vulnerability that led to this attacks are; unauthorized access even when credentials are missing, lack of managing the threat of shared password, failing to ensure

Film analysis Essay Example | Topics and Well Written Essays - 750 words - 8

Film analysis - Essay Example Then, when the seriously ill boy miraculously recovers, sibling rivalry becomes too difficult to manage, and Monica leaves the fake boy in the forest to his own devices. A.I. is enriched with some deep moral themes and issues surrounding motherhood, qualities that make the movie one of the director’s masterpieces (Abu-Mostafa, 2007). The Spielberg work is extremely captivating, and it evokes yet again a stunningly provocative perception of child rearing and of the humanity,  interweaved with a massive feeling of melancholy in the face of science.  The movie draws substantial influence from â€Å"Pinocchio† by Disney. Among the themes that feature in the movie are modernity, false consciousness and consumption.   Ã‚   As Brookfield (2007) as said, modernity of the movie is apparent in its futuristic setting. The beginning of the narrative after the disastrous impacts of global warming helps to reinforce the theme of modernity. Floods that have wreaked havoc in the earth’s surface and uncontrollable population pressures on natural resources are some of the modern challenges that the world will continue to face in the future.  Spielberg predicted a situation where couples will be required to seek for permission to make babies, since the current threats of technological leaps shall have led to the replacement of human beings with mechanical human beings. Interestingly, parents who go for robotic children will have to contend with the fact that those children will remain young and respectful (Abu-Mostafa, 2007). The natural love in the traditional societies will be lost to modernity.   It is arguable that the very fact that â€Å"machine love† has replaced the natural love between individuals serves as the ironic contrast of a movie that attempts to depict hope, but rather in the wider perspective presents an awful representation of social

Rapid Economy Developmentt Essay Example | Topics and Well Written Essays - 1250 words

Rapid Economy Developmentt - Essay Example Both India and China government can now provide timely and more efficient services for everyone’s wellbeing. This is as a result of more people being able to pay their taxes as there are more employment opportunities. The availability of more revenue has thus enabled them to improve on the medical healthcare facilities and education. This not only has private benefit, but a healthy population can be more productive due to the increased life expectancy. There is also the reduction of crime. This is because those lower the social ladder are able to earn enough thus eliminating theft cases. Economic, environmental and global problems that have arisen and are likely to arise On the other hand, rapid growth has caused a lot of harm in China and India. These negative consequences have affected the economic sector, the environment and the global security. These issues arise from expansion of economic activities. As demand keeps on increasing, the more the global side effects. India a nd China being in the beginning phase are experiencing the structural shifts from agriculture to industrial growth. With this, there is a substantial decline in the agricultural share, in these two countries. This has led to the rural- urban migration and thus the amount of farming has reduced. While this happens, the levels of energy consumption go high thus the release of poisonous gases into the environment. To achieve economic growth, these two countries have to increase in manufacturing exports, and decline in manufacturing imports. This leads to increase in energy consumption levels which cause environmental dilapidation. Both China and India are dependent on coal, fuel and electricity as sources of energy. India consumes a lot of energy in the form of solids which include coal and commercial wood. With the rate of economic growth, they had to depend more on liquid fuel, which includes petroleum. However, with the scarcity of oil sources, India had to depend on liquids from th e rest of the world, therefore, started importing, which has peaked annually. China being a popular nation with rapid industrialization requires a lot of energy. From the availability of resources, China depended more on solid fuel mainly coal. However, the energy consumption went higher each year, creating the need for other sources of energy and that's when they started falling back on liquid fuel (Erol & Yu, 1987). By the year 2005, China was the second largest consumer of oil led by United States of America. The net imports for oil seem to be growing each year. This puts an economic strain on citizens who pay for these imports indirectly through their taxes. This high usage of energy in these two countries causes the main problem of pollution. While meeting the high energy demand, the energy production causes the release of toxic gases like carbon dioxide and sulphur. High dependency on coal in both China and India causes severe air pollution by release of sulphur in the air whi ch is the cause of acid rains. The continued emission of these gases is a leading cause of global warming, which causes the erosion of the ozone layer, resulting in the rise, in temperatures. The effects of this are already global; with the melting of snow ice and glaciers, the rivers are overflowing causing high dispensation of waters into the oceans. Other effects of global warming are the increase

Anarchism Essay Example | Topics and Well Written Essays - 3000 words

Anarchism - Essay Example He attended the local school but was primarily self-educated at the town's public library. Proudhon, was among the inventors of socialism, along wih Marx, Bakunin, Blanqui, Blanc, Herzen, Lassalle and Engles. Of these, Proudhon had the profoundest effect upon the workers' movement in the 19th century and his ideas influenced some of the most notable later anarchists, including both Tolstoy and Bakunin, both of whom knew Proudhon personally. Indeed, throughout his life Proudhon acquired and kept a remarkable collection of friends, and as his notoriety spread, acquaintances. Before Proudhon, the word 'anarchist' had been exclusively used as a derogatory epithet to be flung at one's political opponents. Proudhon was the first person to adopt the label with enthusiasm. He denounced the 'government of man by man' as 'oppression,' and in its place advocated a society based on 'equality, law, independence, and proportionality' which 'finds its highest perfection in the union of order with a narchy.' He defined 'anarchy' as 'the absence of a master, of a sovereign,' and envisaged a society in which 'the sovereignty of the will yields to the sovereignty of reason. For Proudhon: " Capital in the political field is analogous to "government". The economic idea of capitalism, the politics of government or of authority and the theological idea of the Church are three identical ideas, linked in various ways. To attack one of them is equivalent to attacking all of them. What capital does to labor, and the State to liberty, the Church does to the spirit. This trinity of absolutism is as baneful in practice as it is in philosophy. The most effective means for oppressing the people would be simultaneously to enslave its body, its will and its reason." ("What is Property", Pierre Proudhon 1840, page 23). One exception to this position was his Proudhon's sexism, causing Joseph Dejacque (as well as subsequent anarchists) to attack Proudhon's support for patriarchy as being inconsiste nt with his anarchist ideas. In his earliest works, Proudhon analyzed the nature and problems of the capitalist economy. While deeply critical of capitalism, he also objected those contemporary socialists who idolized association. In series of commentaries, from "What is Property" (1840) through the posthumously published "Theorie de la properiete" (Theory of Property 1863-64) he declared in turn that "property is theft", "property is impossible", "property is despotism", and "property is freedom". When he said property is theft, he was referring to the landowner or the capitalist who he believed stole the profits from laborers. For Proudhon, the capitalist employee was subordinated, exploited; his permanent condition is one of obedience. In asserting that property is freedom, he was referring not only to the product of an individual's labor, but to the peasant or artisans home and tools of his trade and the income he received by selling his goods. For Proudhon, the only legitimate source of property was labor. Proudhon was remarkably consistent in his thinking about economic issues, but that his rhetoric changed considerably over the years, and that the tactics he adopted in dealing with an understanding of "property" as always somewhat "impossible" shifted slightly. First published in 1840, Proudhon's

Comparing Piaget and Vygotski Essay Example | Topics and Well Written Essays - 1250 words

Comparing Piaget and Vygotski - Essay Example The child learns from adults who behave according to the cultural norms in which the child is reared. The child uses certain adaptive tools for acquiring information from his or her surrounding, language is one such tool (Funderstanding, 2008). Following are some Key Terms of this theory: In Piaget’s approach towards learning, the purpose of education in class room is to provide pupils with opportunities to learn through interaction with the environment. According to Piaget all cognitive learning of a child occurs in stages. This learning is developed by forming of mental concepts. For this it is necessary for the child to have a supportive and rich environment that allows experiential learning. According to Vygotsky, the child’s primary source of education is the teacher’s personality rather than the school environment. The teacher is the child’s MKO i.e. more knowledgeable other and the child internalizes the teachers values and adopts the teachers traits automatically. So the value of education according to Vygotsky is to present the child with an MKO who will be the child’s role model and will transmit knowledge by demonstrating it in front of the child through his or her behavior and actions (Learning Theories, 2010). In Piaget’s approach, the teacher is just a supervisor. He or she just provides the child with an enriched environment, facilitates learning process monitors the child’s work and provides the child with countless opportunities to understand the world and develop his or her own cognitions about the world as the child experiences it. The teacher’s role in this theory is highly objective and impersonal. According to Vygotsky, the teacher is the role model for the child. Whatever the child learns is what he adopts from his teacher. Social cognition is developed in the child by blindly assimilating the teacher’s traits. Intellectual development comes after it. Therefore the role of

The UK post-recession economy Research Paper Example | Topics and Well Written Essays - 4000 words

The UK post-recession economy - Research Paper Example This essay explores the characteristics and attributes of an entrepreneur, examining different theoretical positions about what constitutes entrepreneurial activity and behavior. Once identifying these characteristics, the essay utilizes various examples of how such activities relate to the UK emerging from a recession environment. Opportunity spotting, as an aspect of theoretical entrepreneurial behavior, is known as a type of alertness, schemata giving an entrepreneur the unique ability to create meaning to various market changes that are not achievable with other managers in the business world (Valliere 2013). Such inherent alertness theoretically gives a real entrepreneur that ability to recognize new opportunities where others in society and the business world do not (Valliere). For instance, an entrepreneur may gain access to important statistics about new trends in consumer behaviour and then determine that their business can be adjusted to better service these trends in a fas hion that is innovative and creative, whilst also being designed to satisfy consumer needs in a way not considered by competing companies in an established market. Opportunity spotting, in general, is related to the ability of a theoretical entrepreneur to recognize new market openings or chances to develop a superior business model that can bring a firm greater profitability and competitive advantages by making small-scale or large changes to an existing entrepreneurial venture’s business model.

PHIL 201 Study Guide Essay Example for Free

PHIL 201 Study Guide Essay View and take notes on the presentation, â€Å"An Overview of Issues in Contemporary Justification, Part 1. † What are 3 characteristics of a person’s noetic structure? The sum total of everything that person believes It recognizes the differing degress of certainty, firmness, and conviction with which people hold their beliefs. Characterized by how beliefs are related together. Explain coherentism and the 3 problems with it. Coherentism: All beliefs fall into one category. No beliefs are more foundational than the others. Problems: Circularity: How do I justify my belief in P? It coheres with Q. Isolation Problem: Coherentism isolates my beliefs from the external world. Plurality: It is possible to have 2 coherent systems that are logically incompatible. Read chapter 4 of Epistemology: Becoming Intellectually Virtuous, â€Å"Foundationalism. † As you do, consider the following questions and points: What is the root idea of foundationalism? Each of us holds a set of beliefs basically or immediately while we hold other beliefs non basically or mediately. Why would one adopt a foundationalist approach to justification? Because they are epistemic engies of our noetic structures, imparting to all of our non-basic beliefs, while not themselves requiring justification from any other beliefs – this support goes just one way. What is the regress argument? Taking for granted the general reliability of your conscious faculties, and indeed must take them for granted. What are the 3 basic components of foundationalism? Basic or immediate beliefs- Form the bedrock of what all we believe undergirding everything else we are justified in believing. Mediate or non-basic beliefs- Everything else we believe Basing Relation- Specifies how the epistemic merit of our basic beliefs is to be transferred to out non-basic beliefs. What are the characteristics of strong foundationalism? Specifically explain: The 3 conditions for a basic belief. Must be self-evidently true: those we see to be true immediately, without the benefit of deliberation or argument, merely when we understand the term of the claim in question. Must be Incorrigible: The belief in question is one that is impossible to believe and be mistaken about. Evident to the Senses The means by which basic beliefs support non-basic beliefs. Entailment is the only logical relation that preserves certainty. If one starts with self-evidently true starting points and accepts only what can be validly derived from the same, one thereby insures that one’s entire set of beliefs is untainted and error free. Four â€Å"additional† features of foundationalism. One cannot claim to have knowledge simply by asserting that one knows One must be inwardly cognizant that one’s claims to knowledge is genuine. One must be prepared to show that this is the case. What are the main problems with foundationalism? Note: This section can be challenging; try to understand the main points raised; don’t be concerned about all the details except as a way to understand the following main points: Problems: One’s beliefs in the reliability of one’s senses or memory or consciousness; none of these beliefs is either self evident or incorrigible or evident to the senses, yet they are held as properly basic by virtually everyone. Any acceptance of supposedly pure and certain basic beliefs makes use of various background assumptions or information that compromises their certainty and undermines their basicality. Why only these conditions for a basic belief? Claims that foundationalism solves the regress argument fail (the arbitrariness of stopping points). Other means of supporting beliefs than just deduction/induction (best explanation, retroduction, concurrence, etc. ). The security vs. content problem. The strict demands for four unimpeachable certainty leave one with so small a set of basic beliefs that they can’t possibly bear the heavy weight of all we believe. Other problems (excessive individualism, access requirements, noetic effects of sin). Pg 95. What are the similarities and differences between strong and modest foundationalism? Specifically: What is primae facie certainty? One’s foundational beliefs are not necessarily immune to any conceivable doubt they can be overridden but they are perfectly acceptable unless one has a good reason for thinking they have been undermined. Four of Reid’s contingent truths. The thoughts of which I am conscience are thoughts of a being I call myself. Those things really happened which I distinctly remember. Those things really do exist which we distinctly perceive by our senses, and are what we perceive them to be. What is to be will probably be like what has been in similar circumstances. Reid’s reliabilism of first principles. For our evidence concerning reliability would have to include deliverances of the faculties whose reliability we are accumulating evidence for. On behalf of his foundational principles, they can be correctly identified by their accompanying marks or signs. If someone persists in requesting additional assurances over and above the usual marks accompanying properly based beliefs, then the person holding such beliefs can only respond with genuine bewilderment. Two weaknesses with modest foundationalism. Reid may have underestimated the capacity for thinkers of sound mind and sincere will to disagree about alleged, philosophical first principles. The belief in God can be held in a basic belief. Alvin Plantinga’s belief in God as properly basic. The belief can be justifiably held in the manner of a first principle, that is w/out the benefit of argumentative support. Terms Make sure you can explain the following terms and concepts: Indefeasible- Not able to be lost, annulled, or overturned. Retroduction- A king of reasoning from the best explanation. It is reducible neither to induction or deduction. Concurrence- Observations that cumulate in his belief. Necessary First Principles- They are self-evidently justified, being believed merely upon being understood. Must have these. Contingent First Principles- Not self-evidently justified. Dependent upon something. Doxastic Assumption- A theory that a belief is justified if it coheres with other beliefs.

Stand Up And Speak Out Sociology Essay

Stand Up And Speak Out Sociology Essay What do you do when you know a friend, sister, mother, or child is being abused, mistreated, and neglected? Often times, the answer to this question is nothing. Women from all over are being abused, and no one has a voice to stand up for them. What is the fear of lending a hand, offering shelter, or listening to a story, when the outcome could be a life saved? The violence that young girls and women face goes largely unnoticed by a vast majority of people within society, and small numbers stand up for the cause to fight the abuse that females endure. Women around the world are still being treated unfairly, the justice system has failed them, and they are controlled by a mechanism called fear. I have been doing a lot of research on violence against women, and I have had a very close encounter with a type of violence that women face every day. Through the numerous amounts of research, personal testimony ¿Ã‚ ½s, and videos, I have realized that violence against women only has one requirement: being female. Violence against women does not focus on black or white, rich or poor, young or old, or even married or single. According to the National Organization for Women, Domestic Violence is defined as a pattern of abusive behavior in any relationship that is used by one partner to gain or maintain power and control over an intimate partner. There are many different forms of abuse that women and girls can suffer from, and stepping up is the only way to stop it. There are many different types of abuse that women endure that range from verbal abuse to physical abuse. However, one of the most prominent types of abuse is physical violence. Many women from across the world are controlled by physical violence inflicted by men, and even in some cases women. A Safe Place has reported that about every nine seconds, a woman is physically abused by her husband. The World Health Organization has reported that between 16% and 52% of women have been assaulted by an intimate partner. In a recent article that I read about battered wife who kill, this woman ¿Ã‚ ½s husband would beat her for bills coming at the same time, forgetting to pick up something from the store or even giving the wrong answer to a question that he asked. A Safe Place also mentions that 95% of assaults on spouses or ex-spouses are committed by men against women. The women that are being physically abused are dealing with being beaten with objects, burnt with cigarettes, being punched , getting kicked, and even being spit on. The abusers in the cases stop at nothing to harm the intimate partners. Many people that abuse in intimate relationships, are just seeking to be in control of the relationship. It is stated in Gendering Violence,  ¿Ã‚ ½For feminists, power and control are the core of male violence ¿Ã‚ ½ (McLeod, and Gilgun 167-193). We live in a world where gender roles are still embedded in a vast majority of people ¿Ã‚ ½s thoughts and minds. A women ¿Ã‚ ½s work is supposed to be cooking, cleaning, and taking care of the children. In addition to this, women are expected to be passive and obedient to the every command of her husband or intimate other. This control mechanism that men strive so hard to have, is why many women are abused to date. This is typically referred to as hegemonic masculinity, which according to Wikepedia refers to the belief in the existence of a culturally normative ideal of male behavior. In Gramsci ¿Ã‚ ½s Prison Notebooks, hegemony as originally formulated is about the winning and holding of power (McLeod and Gilgun 167-193). Power and control is what the integrity of a woman is worth to some men. A very controversial topic of violence against women in intimate relationships is marital rape. Some men believe that women have to always be willing and ready to perform sexual acts, and some women believe that it is not rape as well. Being woken out of your sleep, being touched when you don ¿Ã‚ ½t want to, and being forced into sexual activity with a partner all fall under the category of rape. I have read several articles where women are sleeping with the person that they are in a relationship with and awaken out of their sleep to a body on top of them or wandering hands. It seemed that it took to be in the situation to actually feel how these women did, but the women in the articles felt violated just as they would if it was a stranger. In the National Violence Against women survey, approximately 25% of women said they were raped and/or physically assaulted by a current or former spouse, cohabiting partner, or date in their lifetimes. Rape itself, which has been around for age ¿Ã‚ ½s, dates back to slave days. People of Caucasian backgrounds would rape black women to enforce control over the black men. It left them hopeless, with a feeling that they could not do anything to protect their women. Even to this day, rape is a major problem for women and young girls. According to the National Crime Victimization Survey, which includes unreported rapes to the police, 232,960 women in the U.S. were raped or sexually assaulted in 2006 (National Organization for Women). Rapes are rarely reported; in fact the FBI estimates that only 37% of all rapes are reported to the police. Another disturbing fact is that in 2000, nearly 88,000 children in the United States experienced sexual abuse. (ACF 2002). Not even our children are safe, which is who we strive to protect and keep out of harms way each day. From testimonies that I have overheard, majority of the child sexual abusers are family members, and sometimes the father of t he child. The women that I heard these stories from were adults, where the sexual abuse occurred years ago. The abuse that these women suffered as children continues to haunt them many years later. These child abusers don ¿Ã‚ ½t realize the scar that they are leaving on the women and young girls that they are violating. A common thread in all of the different types of violence that I have previously introduced, are all controlled by fear. Fear is what feeds the abusive nature of these men and women, who are inflicting abuse among women and girls. Fear has caused women not to come forward in reporting what is going on with the abuse in their households. Women fear their safety and the safety of their children. Women fear being left alone with no resources, money, or family to assist with the leaving process. Women also fear being embarrassed or turned away from family or friends because of what they have experienced. According the to Bureau of Justice Statistics, only 36% of rapes, 34% of attempted rapes, and 26% of sexual assaults were reported to the police in 1992-2000 (Renninson). I go to say that women are still treated unequal because of the multiple variations of abuse that women endure every day. Not only does a significant other abuse some women; they are also abused by a justice system that is solely based around the hegemonic male figure that I have previously mentioned. When a father sexually abuses his child, the blame for this occurring is placed on a mother for not protecting her child. The blame is therefore taken off of the perpetrator, and is instead put onto another one of his victims. Since mothers are supposed to nurture and protect, it is not in the norm of our society for anything to happen to our children. I have recently watched a video where children have come forward and admitted that their father was sexually abusing them, and custody was ripped from these mothers. I ¿Ã‚ ½m not talking about unproven or undocumented allegations, these children have seen plenty of doctors who have verified with courts that misconduct was going on. But because court systems are based around men, the thought of a father wanting to take care of his children ultimately deems him the fit parent. Judges didn ¿Ã‚ ½t care one bit about what these children were going through, because fathers just wouldn ¿Ã‚ ½t behave in this manner. In Regulating Womanhood, it was stated  ¿Ã‚ ½children tend to be safest from sexual abuse in the absence of men, but it is in part men ¿Ã‚ ½s separation from children which makes them a threat when they return ¿Ã‚ ½ (Regulating Womanhood: Historical Essays on Marriage, Motherhood and Sexuality 53-77). A mother is expected to keep the child away from the abusive situation no matter what her stability situation may be. Another injustice that women are facing is a lack of protection, to assist them in leaving a bad situation. If you are a prostitute, lesbian, handicapped, or deaf, your protection services are pretty much limited. Shelters don ¿Ã‚ ½t have the proper equipment to assist deaf or handicapped victimized women, and they turn away lesbians and prostitute. Does protection from a violent situation come with a face, a sexual preference, or a completely functioning body? If we cant attempt to protect every kind of women being victimized what are we really doing? Doesn ¿Ã‚ ½t every women deserve to be treated with the same respect and dignity as the next? Women are scared to leave an abusive situation when the reality is that they may have no money, no food, or a warm place to sleep at night. So turning away minority women is hurting us as a whole, not just as individuals. Have you ever jaywalked, played your music too loud, or parked your car on a street all night? The offenses just named have the equivalent charge as punching your girlfriend in the face or breaking your wife ¿Ã‚ ½s ribs. What does that say to you? Speaks of one of the most injustices that plenty of women face everyday. Cheryl Winters recited,  ¿Ã‚ ½ It is hard for abused women to find justice, you see, when our lives hold the same value as someone found guilty of vagrancy ¿Ã‚ ½. If abusing your significant other holds the same consequences as a misdemeanor, what does that speak to men but of the minor value of a woman? Things implemented to help protect women, are actually benefiting men help get around the system. I ¿Ã‚ ½m sure once or twice you have come across stories or headlines about battered women who kill. These are the women who saw no other way out than the kill their intimate partners. These women feel that this is the night that he is going to kill me. This is when the mentality of  ¿Ã‚ ½rather him than me ¿Ã‚ ½ mentality begins to kick in. The opinion of certain men about the dominant role that is associated with the hegemonic role is so disturbing. William Blackstone many years ago stated  ¿Ã‚ ½ if the baron kills his feme it is the same as if he had killed a stranger or any other person; but if the feme kills her baron, it is regarded by the laws as a much more atrocious crime, as she not only breaks through the restraints of humanity and conjugal affection, but throws off all subjection to the authority of her husband ¿Ã‚ ½ (Schneider). This again results back to the control that man is supposedly entitled to, and it shows how strong the belief was in the dominance of man. Main arguments in women who kill their partners are battered women syndrome and or self-defense. Schneider stated that work on self-defense for battered women who kill has been premised on the notion that self-defense requirements of reasonableness, imminent danger, and equal force are sex biased. I completely agree with this statement seeing as equal force between a man and woman is highly unlikely and the fact that a woman is acting out of the norm of a woman ¿Ã‚ ½s traditional role is unreasonable. In plenty articles and movies that I have viewed and read, women are getting charged more harsh and men more lenient. Actually Schneider has an article where a man got 18 months in a work release program for killing his wife after catching her sleeping with another man. The judge stated that  ¿Ã‚ ½ he could only imagine nothing that would provoke an uncontrollable rage greater than this: for someone who is happily married to be betrayed in your personal life, when you ¿Ã‚ ½re out working to support the spouse ¿Ã‚ ½ (Schneider 117). Here we see once again an exception for a man to violate his intimate partner, even if it results in killing her. Since a woman is supposed to accept openly that her partner is cheating on her, for her to act in killing is atrocious, but for a man to act in the heat of the moment is absolutely normal. What ¿Ã‚ ½s acceptable for a man is of course unacceptable for a woman. I have learned that more needs to be done to protect the women in our country. The things we value and believe in need to be change to fit the new way of living. It has become clear to me, that men value money, control, and structure more than they value of women. We need to do more to continue and widen protection for women. We need to learn how to stand in, step up, and speak out!

The Changing Business Environment Of Samsung Marketing Essay

The Changing Business Environment Of Samsung Marketing Essay This is summary for Changing Business Environment of Samsung over last Five Year which will give an idea about why it is important for any Trading or Manufacturing organization to understand the business environment (Macro Micro) i.e. to sustain and grow over long term with the help of developing different strategies. This report briefly shows the history of Samsung which identify its nature diversity of business over last few years. This report include usage of ( SWOT, PESTEL, BOSTON BOX MATRIX Analysis etc) to evaluate and understand the changing environment of Samsung justifying benefits of using such Analysis tools. In evidence couple of graphs has been presented to show companies performance in global market. It shows the sales figure/Financial statement for the past FIVE years which helps in understanding how company is performing in different regions/countries. This report comprises of conclusions based on findings and also in general recommendations for the topic. Introduction: Samsung has grown to one of largest electronics company since 1938. Since 1970s and early 1980s Samsung expanded globally with diversified intention into core technical business. Samsung leads the electronic industry with its high performance and with high growth rate along with stability. 2009 Global Market Share of 13 Percentage of different products available in market. Examples -: Mobile Phones, Monitors, Semiconductor. 1970 First Black White (Model P-3202) production started Samsung-Sanyo Electronics. 1969 SAMSUNG-Sanyo Electronics established (renamed SAMSUNG Electro-Mechanics in March 1975 and merged with SAMSUNG Electronics in March 1977) 1951 SAMSUNG Moolsan established (now SAMSUNG Corporation) 1938 SAMSUNG was founded in, Korea (Taegu) Company: Samsung Global Market share is terms of sales is as follows: TV Market Share is 21.9% Laser Printer Share is 13.7% Mobile Phone Share is 16.7% DRAM Share is 30.1% TFT-LCD Panel Share is 25.7% In the above graphical presentation we can see the performance of Samsung in various sectors of Electronics and Telecommunication. If we compare this performance to other companies Samsung emerges as world leader in at least 3 sectors which are TV, DRAM TFT-LCD. This clearly shows us the dominance of Samsung in market and this is mainly due to its transparent policies, positive approach and honest efforts in being a global leader. This performance data is taken from Samsungs sustainability report for 2009 from Samsung official website. Unit KRW trillion Sales 2006 2007 2008 2009 2010 Korea 19.3 21.1 23.6 24.2 N.A America 18.0 19.6 25.4 26.0 N.A Europe 22.0 27.0 34.2 34.0 N.A Asia 14.8 15.7 19.2 23.2 N.A China 11.8 15.1 18.9 20.0 N.A The above data shows the sales performance for the past 3 years in regions across the globe. We can clearly see that Samsung as a company is growing locally and globally Korea being its local market. This consistent growth of the company is mainly due to its marketing strategies which are paying off well. More details on Samsungs operations, marketing, product line etc can be found on : http://www.samsung.com/uk/aboutsamsung/citizenship/oursustainabilityreports.html http://www.ameinfo.com/178504.html The business environment keeps on changing and is unpredictable. So, it is very necessary to understand and react to this changing business so as to survive and grow in the market.Proper knowledge of business environment will make the entrepreneurs and businesses able enough to plan and implement strategies that are needed to identify the scope of improvement, create and exploit opportunities that come on the way, retain stability, gain competitive advantage and prepare appropriately for the upcoming challenges. The way the environment is analyzed and accordingly the strategies implemented, will define the track of the business.Here With the example of samsung. The data collected will be analysed using SWOT. SWOT stands for strengths, weaknesses, opportunities and threats. SWOT analysis, is a strategic business planning tool used to determine the Strengths, Weaknesses, Opportunities, and Threats that exists in business. It includes identifying the business objectives as well as the internal and external factors which are likely to favour the business. It may be classified into: Internal factors strengths and weaknesses that are within (internal) organization. External factors opportunities and threats of external environment. The internal factors can be classified into strengths and weaknesses depending upon their effect on organization and business objectives.The factors may include all of the 4Ps; as well as personnel, finance, manufacturing capabilities, and so on. The external factors may include macro-economic factors like technological , legal,and socio-cultural changes, as well as changes in the marketplace or competitive position. According to B Kyle SWOT can be identified as internal and external in relation to environment. Strengths: It usually tells about the positives of the company. How well we are doing in our area of interest. What is our position in front of competitor? Weaknesses: Weakness refers to area of struggle of our company. Why are consumers not happy about particular product? Why are we not meeting sales target for a particular product. Opportunities: It is directly related to external environment. It could be the areas where your competitor is not good at. We should try to utilize strengths in areas of our weaknesses. Are there any emerging trends or possibility to capture market by introducing new product? Threats: It could be financial or development problem. Competitors strength is major threat. Are competitors becoming strong. Description. Strategic business tools: SWOT analysis for Samsung Strengths Strong hold in Consumer Electronics Low margin strategy increases sales Highly skilled labour availability locally globally Good focus on customer service and good marketing strategy Strong management and loyal employees Weaknesses Lack of performance in Laser printer sales Less cost effective in PC consumable products. Lack of brand recognition in mobile phone sector. Poor pricing strategy Lack of usage of infrastructure in some part of world e.g. China Opportunities Can unseat major stake holders in TV and DRAM sectors Increase product range in consumer electronics Can sustain future market using the idea of consumer taste research Rapid growth in competitive markets across the globe Increase in portfolio due to acquisitions of other firms Threats Faces high competition from Sony and Nokia in mobile phone sector. Daily entrants of new technology companies Impact on sales due to recession Lack of innovation in mobile industry threatens its position further down. Intense competition from existing companies in PC sector. The above information which helps in doing the SWOT analysis and other graphical presentation helps to answer our objective questions mostly. Although it might not provide direct answer but clarifies strategies and benefits of organisation doing global and domestic business. It clearly reveals the benefits of proper marketing in terms of sales numbers. PESTEL Analysis  : PESTEL analysis is one of the most important tool of business strategic management which helps in identifying the surroundings within which the company operates. It gives a clear idea about the threats, risks and opportunities that occur in the market. It helps in understanding the needs of market, its fluctuations, whether the market is progressing upwards or downwards (i.e. growth decline ratios) and thus makes the businesses able to develop strategies accordingly. PESTEL is used as decision making tools in macro-environment. PESTEL is Classified into :- Political factors Economic factors Social factors Technological factors Environmental factors Legal factors Political factors :- Its all about intervenes of the government on the economy of the country. Political factors include areas like Tax policy Labour law Environmental law Trade restriction Tariffs Political stability Services and goods provide by the government ( merit goods ) and services which is not provided by the government ( demerit goods ) Goodwill among foreign nations Economic factors :- It includes Interest rates Economic growth Rate of inflation (Inflation rate) Rate of Exchange (Exchange rate) For example :- Cost of capital is directly affected by interest rate Exporting goods and supply of goods is affected by exchange rate Inflation raises cost. Social factors :- Demand of products and company operations are affected by social factors like Culture of the society Natality rate Growth rate Age distribution Technological factors It includes Research and development activity. Automation Better technology for the production of goods Technological have direct impact on costs, quality which leads to innovation. Environmental factors It includes- Weather Climate change Locality (Place) Ecofriendly products (to overcome with problem of global warming) e.g Effect of temperature on farming and tourism. 6. Legal Factors It include Consumer law Employment law Health and safety law Discrimination law e.g Minimum wages system in U.K is legal factor that affect business. Minimum age for employment is fixed by the government. Pestle analysis For Samsung Boston Box matrix revive Conclusions Recommendations We have analysed and gathered data from Samsung Company in relation to our topic global versus domestic marketing a critical analysis. The conclusions drawn using company data alone cannot summarize our findings. However there are similarities in few points. If we look at the organisation section we can see how Samsung has come a long way to be recognised as a global brand. It shows that achieving such position in global market is very hard. Not only achieving but it becomes increasingly important to sustain that growth due to fierce competition. It shows how companies have to adjust or change their policies from time to time keeping in mind rising and falling economy. In general it alerts companies of being more innovative cautious of environmental changes. It also emphasis on limitations one has to face as the behaviour and taste of consumer changes geographically. Recommendations Companies should develop marketing strategies keeping in mind its strengths, weaknesses and availability of resources to be globally successful. Companies should try to develop product which global consumer can identify easily from competitors one. Identify a sector where we can see growth and sustain competition. Companies should appoint special taskforce or focused group people to identify the drawbacks of non performing sector and find solutions to that. To be successful organisations should concentrate mainly on quality and pricing structure. Be in constant touch with customer by means of advertising, press releases and make them aware about product specifications and changes if there are any. Try to use both forms of communication verbal and non verbal. Summary From this report on global marketing versus domestic marketing we understand that main objective of any company doing global marketing is to find opportunities. It mainly expands companys reach to global customers. This mainly depends on how the company analyses its strengths and opportunities, optimum usage of resources and approach towards business. Here in this report we saw how Samsung as a global company has wide range of products and constantly keeps on adding them to sustain market place. SWOT analysis helped us to understand about positives and negatives of the company. Important outcome of the report about marketing can be converted in one slogan think globally and act locally.

Human Cloning is not a Moral Issue Essay -- Argumentative Persuasive T

Human Cloning is not a Moral Issue There has been an onslaught of protests and people lobbying for a ban on human cloning ever since Dolly the sheep was cloned by Dr. Wilmut and his colleagues. Not only has the media exploited the ethics of the issue and scared the public with images of Frankenstein and evil scientists, but many worldwide authorities such as President Bill Clinton, Jacques Chirac of France, and Prime Minister John Major of Great Britain have all spoken out publicly against human cloning and have proposed legislation and committees to regulate and/or ban it. Religious leaders and the Pope have also talked about human cloning's dangers of "Playing God" and the "loss of uniqueness" that an individual would suffer if he or she were to be cloned. Many of these fears are misguided, misrepresented, and illogical. The benefits of human cloning would far outweigh the drawbacks, and cloning research should not lose funding or be banned. The most popular objection to human cloning is the assumption that science would be playing God if it was to create human clones. Reverend Albert Moraczewski states that cloning is intrinsically morally wrong, is an attempt to play God, and exceeds the limits of the delegated dominion given to the human race. (Madigan, 8) To say that this argument is unclear and unsupported is generous. It refuses to differentiate between cloning and the interruptions of biological processes which are viewed as commonplace in today's society. Religious objections were once raised at the prospects of autopsies , anesthesia, artificial insemination, organ transplants, and other acts which were seen to be tampering with divine will. (Madigan, 3) Yet enormous benefits have been reaped by each of these... ...crime or illegal activity has a victim involved with it. Human cloning has no victim or loser in it. It is a chance for the advancement of science and humankind. Banning it or eliminating research funds would be a tremendous mistake when we haven't even perfected it or discovered all of the possible benefits. Works Cited Hopkins, Patrick D. Bad Copies: How Popular Media Represent Cloning as an Ethical Problem. Hastings Center Report April 1998: 1. SIRS Researcher. West High Library, Iowa City. 25 Jan. 2000 Madigan, Timothy J. Cloning Humans. Free Inquiry Summer 1997: 1. SIRS Researcher. West High Library, Iowa City. 20 Jan. 2000 The benefits of Human Cloning. Human Cloning Foundation. 25 Jan. 2000 Vere, Stephen. The Case for Cloning Humans. Taking Sides: Science, Technology, and Society. T. Easton (ed.); McGraw-Hill Publishing, 2000. 1-12.

Interesting Person :: essays research papers

An interesting person should have lots of intrigues in his brain, and he should have a curious characteristic. So the adjective "interesting" seems to be the best word to describe me, there is no other better word. All of my old friends know I am a person who has lots of intrigues and happiness. In every important party, I am always invited to be the joker. For these reasons, I believe that I am the most interesting person I know. Interesting things were not strange to me when I was young. I still remember when I in primary school, I always thought about the difference between boys and girls. The most interesting matter was that why boys and girls went to different restrooms, and what was the difference of the 'equipment' inside restrooms. This question was always running around in my brain. Once I asked my father about it, but it might because my father grew up in a traditional Chinese family in which no one could talk about sex, his answer was " you will know when you get older!" which made me frustrated. Because I was extremely interested in this matter, so once I decided to switch the male-restroom card and the female-restroom card to see what would happen. The consequence was that no one knew how to use the 'equipment' in the switched restrooms. Finally, my class teacher who blamed me for tricking classmates told me about the differences between boys and girls. Till I entered high school, I still did not change my naughty characteristic. I have a very interesting experience to share with you: One time my brother drove me to school by his new Honda sports car, he drove faster by my request as I was going to be late. He stepped on the gas deeply and the sports car activated its V-tech function, in not more than 5 seconds, the sports car reached sixty miles per hour on the road supposed to drive thirty-five miles per hour at most. Unfortunately, we was unknowingly caught in an automated speed trap hid somewhere on the road, and it measured our speed using radar and photographed our car. Two days later, I received a ticket for forty dollars and a photo of my brother's car. Instead of payment, I sent the police department a photograph of forty dollars. Several days later, my brother told me that he received a strange letter from the police department that contained a ticket for a hundred dollars and a photo of handcuffs.

Life of Cleopatra

One of the well thought-out classic scopes was the life of Cleopatra. She was considered as one of the most famous and influential figures in olden times. Because of the Hollywood film done for her account it was very tough to find which was real and which myth was. Her life was full of scheme and cleverness, and mystery. She was truly a dominant and radiant woman whose places in history, her tools that took her to manage many powerful men in her life and also led her to ultimate demise were her beauty, native allure and appeal.A lover, warrior, mother, enthusiast of all that brought splendor, queen of Nile, and were the account associated to her, but the certain thing was that she was last Pharaoh of Egypt. The life of Cleopatra was influenced the mythological ideas of the most writers as well as Shakespeare. Through her life the substitute ability to Catholic academic doctrine was recognized. As the matter of fact the influence of outside literature and arts were commanding in Rome up to now. And by her impressive history, many writer was trying to dig deeper the reality of her life.And this was the beginning of her epic life. Cleopatra VII Philopator or Cleopatra the destined queen was born in Alexandra the former capital of Egypt 69 years before the birth of Christ. A beautiful baby was the eldest child of Cleopatra VI Tryphaina and Ptolemy XII Auletes Theos Philopator Philadelphos Neo Dionysos also known as Ptolemy XII the reigning pharaoh of that time. At the very young age of nine, Ptolemy her father was allegedly a son of a non-married couple, her father was trying to overcome the trial for his fading supremacy by bribing authoritative Romans.Cleopatra VI was died when they returned to Egypt. Through Roman forces the throne was regained by his father. At 51 BC her 9 years old brother Ptolemy XIII was married to her at the age 18, with the same age together with her brother and husband she became the queen of Egypt by inheriting the ruler ship of her fat her. In the beginning of Cleopatra’s reign all was not so easy due to the fact of her age, many groups were too envious of her cleverness and for ruling the entire Egypt unaided and she had to compete with her enemies, and these were the reasons why she struggled completely.The Romans were conquering city after city, until the ministers pressed Cleopatra out of her command after ruling the Egypt for three years. She moved out of Egypt and lived in Syria and left the throne to her brother alone that she thought agreed with the situation. Because of the circumstances she was too infuriated but didn’t give up. When she learned that Julius Caesar was in Alexandria, Cleopatra planned to go back to her native land in secret to escape from the hand of her brother.To get coalition against her brother Ptolemy XIII and to defend her mother landed from fraud, as what the most accounts told that Cleopatra offered her virgin body and surrendered it in the form of rug, she covered u p herself in an oriental rug and presented it to the general who almost four times of her age . And she got the attention of the Romans general, Julius Caesar fall in love at the first sight to her when the rugs unfold. Her charisma and allure beauty surpassed the influence of Caesar. Ptolemy XIII affirmed warfare against Caesar and his Roman soldiers since he believed that he was deceived.Caesar took controlled of the throne of Alexandria when he defeated Ptolemy XIII in the war. After the battle Caesar due to his loved to Cleopatra he gave back the power to Cleopatra to rule the Egypt alongside with her brother Ptolemy XIV as co-leader. In June 47 BC Cleopatra bore a child and to emphasize that her son was from Caesar she named him Ptolemy XV Caesarion, but Caesar never properly acknowledged fatherhood. In 46 BC General Julius Caesar returned to Rome he brought Cleopatra and his son showing as a prisoner of war because he was already married with Calpurnia.But Cleopatra fought for her privilege; she asserted to be the wife of Caesar that brought him to assassination in 44 BC. After Caesar murdered Cleopatra go back to Egypt, she recognized her son Ptolemy XV Caesarion as her co-monarch regardless of the rumor that she perhaps killed her brother Ptolemy XIV. Rome Empire began to divide upon the death of Julius Caesar, and because of Caesar assassination Cleopatra’s throne was in danger; she collaborated with Marc Antony the subsequently Roman forces governor of the area, an old companion of Caesar.She proved to Marc dramatically and convinced him that she was not guilty about the allegation to her regarding her support to the followers of Julius in Rome, apparently, she got the hold up and captured his interest. Marc left Cleopatra with twin in her womb after he spent the whole winter in Alexandria with her. In the meantime he went to his first wife Fulvia who died in 40 BC. In 37 BC Marc went back to Antioch with Cleopatra and undergone a matrimony of marriage in 36 BC. With the same year a baby boy named Ptolemy Philadelphus was born to them.After they knew that Ptolemy XV was lost controlled his territory including Lebanon which is formerly part of Cyprus, officially Marc renovate to Egypt. After the military victory in 34 BC he tied Cleopatra in Alexandria and recognized Caesarion as the son of Julius Caesar and affirmed the joint rulership of Cleopatra and her son. When the Romans threatened specially Octavian formerly ally of Antony, conceding of country by Marc to Cleopatra was used by Octavian to destroy the trust of the ministers to him.Because of this, the Romans were not happy to their relationship because they considered that the loyalty of Marc was not in the Rome Empire anymore since he gave away the power of ruling Egypt to Cleopatra. In 31 BC Marc Antony lost to Octavian in Battle of Actium because of the misstep pointing to Cleopatra. Cleopatra tried to persuade Octavian to support her offspring to progression of authority but she was unsuccessful to make a deal. Unfortunately, luck was twisted against them. After the defeat of Marc Antony to Octavian and lost all his support he committed suicide and die in the arm of his beautiful wife Cleopatra.Since all of the Roman forces go against Cleopatra she decided to cut off her life by poisoning herself with the use of snake and died in admiration and was the very last Pharaoh of EgyptBIBLIOGRAPHYAnalysis of Four Character of Shakespeare http://www. free-essays. us/dbase/d3/lva130. shtmlAncient Egyptian Pharaohs: http://www. 123helpme. com/view. asp? id=23405Biography and history of Cleopatra http://en. oboulo. com/biography-and-history-of-cleopatra-44856. htmlBiography of Cleopatra http://www. chevroncars. com/learn/famous-people/cleopatraChronology of Cleopatra’s Life http://atrium-media. com/bibliotheca/romanhistory/cleopatrahist. htmlCleopatra – Queen Of Egypt: http://www. 123helpme. com/view. asp? id=58913Cleopatra, Ruler of E gypt http://www. infoplease. com/biography/var/cleopatra. htmlCleopatra’s Wiles http://www. azete. com/preview/58914Greco-Roman Influence in Shakespeare's Antony and Cleopatra http://www. 123HelpMe. com/view. asp? id=16285Isabelle Vignier. The Tragic in Antony and Cleopatra, June 2004 http://www. literature-study-online. com/essays/antony-cleopatra. htmlShakespeare’s Antony and Cleopatra http://www. englishessays.org. uk/english-essays/shakespeare-anthony-cleopatra. phpThe Female Pharaoh Cleopatra – History Essay http://www. freeonlineresearchpapers. com/female-pharaoh-cleopatraThe Love Story of Antony and Cleopatra http://www. azete. com/preview/8726The Masks of Anthony and Cleopatra http://www. thefreelibrary. com/The+Masks+of+Anthony+and+Cleopatra-a0161065564The Story of Cleopatra in Ancient Egypt http://www. echeat. com/essay. php? t=30239What are some conflicts and compromises in Cleopatra's life time? http://answers. yahoo. com/question/index? qid=20071227 084535AAGJBCP

Titration Journal

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