Columbus vs. de Vaca Essay examples - 698 Words

Christopher Columbus and Alvez Nunez Cabeza de Vaca were both explorers for Spain, but under different rulers and different times. The more famous, Christopher Columbus, came before de Vacas time. Columbus sailed a series of four voyages between 1492 and 1504 in search for a route to Asia which led accidentally to his discovery of new land inhabited with Indians. Christopher sailed under the Spanish monarchs, Ferdinand and Isabella for his journey to the Indies, whom he was loyal to by claiming everything in their name. De Vaca , followed in Christophers footsteps and journeyed to Hispanionola for Spains emperor, Charlves V, the grandson of Ferdinand and Isabella. Both, Columbus and de Vaca composed a series of letters addressing†¦show more content†¦His sole purpose was to inform others (of his sufferings and his discoveries of the Native Americans). He also wanted to justify his conclusions regarding Spanish policy and behavior in America which is mainly addressed to Ch arles V. De Vaca believes that [his] only remaining duty is to transmit what [he] saw and heard in the nine years [he] wandered lost and miserable over many remote lands. Therefore, he conveys to Charles V the many incidents that occurred throughout his struggle for survival while in Texas. In De Vacas opinion, he thinks that the information he is revealing will be useful to others and will be of no trivial value for those who go in [his majestys] name to subdue countries. The descriptions which Christopher Columbus and Alvez de Vaca reveal are entirely different. Columbus wrote information that was insignificant. His explanations are very vague and are only somewhat in depth when something interests him greatly, like his discovery of the beautiful Espanola. Columbus wrote about the Indians and their land as if they were nothing of importance. The majority of his descriptions of explorations were about himself or based on himself. On the other hand, Alvez de Vaca claims that he is telling the truth and are strictly factual. De Vaca remembers all the particulars, in other words, every significant detail. Alvez mentions both positive and negative qualities of his experiences. It seems as if heShow MoreRelated Christopher Columbus vs. Alvez Nunez Cabeza de Vaca Essay677 Words   |  3 PagesChristopher Columbus and Alvez Nunez Cabeza de Vaca were both explorers for Spain, but under different rulers and different times. The more famous, Christopher Columbus, came before de Vaca’s time. Columbus sailed a series of four voyages between 1492 and 1504 in search for a route to Asia which led accidentally to his discovery of new land inhabited with Indians. Christopher sailed under the Spanish monarchs, Ferdinand and Isabella for his journey to the â€Å"Indies,† whom he was loyal to by claiming

International Trade Law Free Essays

Law chosen to govern a transactions is clearly state the legal consequences of their contractual activities for example the right, obligation, and remedies for involve parties, and they can choose the law of particular country or international law to govern their contract. International trade law (CISG) includes the appropriate rules and customs for handling trade between states and it forms part of domestic law if the involve parties are from the contracting state of CISG. With assistance from Unification of Private Law (UNIDROIT) for filling gap in the coverage of issues by the CISG which is the validity of contract, effect of contract on property and goods, exclusively or non-sale aspects for distribution agreement, and inability of sell for death or personal injury cause by the goods on any person. We will write a custom essay sample on International Trade Law or any similar topic only for you Order Now The domestic law that governs the transactions in Malaysia is the Contract Act 1950 and supplement from Sale of Goods Act (SOGA) 1957 (revised 1989) which is based on the English Sales of Goods Act. As a Malaysian lawyer, I recommend you choose the Contract Act 1950 and SOGA as the governing law because the business you based is on Malaysia home soil and it creates a familiar factor to you. Besides that, Contract Act 1950 and SOGA already govern the basic contract of goods and contract of insurance but they did not cover the contract of carriage. However, because of Malaysia still practices the Hague Rules by virtue of the Carriage of Goods By Sea Act 1950 (Revised 1994), you have to choose the Hague Rules to govern your contract of carriage even though there are prominent weaknesses. For contract of carriage, there is standard term used on trading call as International Commercial Terms (INCOTERMS), and Cost, Insurance and Freight (CIF) and Free On Board (FOB) are the generally used term in the trade. So, I recommend you to practice FOB even through your product price will slightly lower due to bargain from buyer, but the cost will reflect on save at the transport of the products. Besides, the main benefit is you do not need to make arrangement on carriage and thus this will reduced the burden to you as a seller’s responsibilities. Policies and regulations have the very close relationship because regulations are come under the policies. The policies and regulations at Malaysia are based on an open and encourage motive, so, normally you can smoothly doing your business on export the product out of Malaysia to foreign countries. This is see through the durian is one of the fruits that identifies by the Third National Agricultural Policy (1998-2010) (NAP3) as important role in creating competitiveness of the Malaysia fruit and vegetable industry in the ASEAN. However, you need to take care about different policies and regulations of your dealing countries which are ASEAN countries and China in order to gain the benefits from all your dealing exporter countries which are actually on the free trade area as ASEAN Free Trade Area (AFTA) and also ASEAN-China Free Trade Area (ACFTA). Firstly, other than the list of preferential tariffs products that under the Common Effective Preferential Tariff (CEPT) scheme , the 40% rules of origin are also one regulation that need to comply with in able to benefit from preferential market access. So, you need to obtain a different certificate of origin from Ministry of International Trade and Industry (MITI) to trade at both free trade areas. Besides certificate of origin, there are regulations for the quality of trading goods on AFTA and ACFTA. Start from sign of AFTA and ACFTA, the ASEAN countries and China fruits market move to more open market as can see through the fruits quality control have been replace to which is more harmonize and standardize call as Sanitary and Phytosanitary Measurers (SPS). This is to prevent countries to protect their domestic agricultural producers from imports with stringent phytosanitary measures which are non-science based, discriminatory and non-transparent. So, you now can be more efficiently and effectively on export your product to these particular countries. ? Answer 2: International agreement different to domestic contract that only contracting within the familiar home country itself, it is more complicated in contracting with various countries and sometimes may be in unfamiliar countries. So in contracting International agreement, there normally required for more trade documents that covers wider range that classified under four main groups which is Financial, Commercial, official, and Transport and insurance documents. Compared to International agreement, domestic contract normally required fewer types of documents especially only commercial and insurance type. This is because domestic contract only contracting the goods move within country territory and did not cross over he national boarder, so documents like certificated of origin in Official group of documents, bill of landing (BOL) or airway bills (AWB) in Transport group of documents are not needed. Term of payment decide on International agreement is more complicated than for domestic contract in reasons of more person involve in the payment process for International agreement. This process can explained though the general example of payment term which is letter of credit (LC) that involve bank parties assista nce by act as a middle man in the payment process. The next main difference between both is the risk face by each other. International agreement is exposed to a number of risks such as buyer’s risk, transport risk and transfer risk that may be also faced by domestic contract. However, these similar risks faced by the domestic contract will be lower in term of cost factor and some other risks such as exchange rate risk and country or sovereign risk will exclude to domestic contract that only contracting at local currency and local policies. Besides that, the transportation and delivery aspect must follow the international standard for example the standardized dimensions of shipping pallets for International agreement, but this requirement is not so strict for the domestic contract. Product packaging and labeling aspect is also not so concern by domestic contract because it normally travels across short distance. However, for International agreement that the goods travel at long distance, export packaging must be suitable for the particular mode of transport in order to provide maximum protection. There are four different types of contracting methods available which is negotiating a complete contract, choosing international law to govern the contract, agree on standard form or terms, and standard industry contracts. Negotiating a complete contract is not suitable to you because your business was just at the beginning stages of entering the new market, so there are many ‘unknown’ on the others’ domestic law that will cause unfair situation in the contracting, thus this will also incurred even more time in making the final agreement. For your situation that deals with many countries, standard industry contracts seem more suitable to you but there are still not any single association that published the standard contracts of durian even though there are already mature grow of durian industry in ASEAN. Then, Standard terms contracting method is suitable to you not only because it is a speedy and convenient way of contracting, but it also benefit to you as an fferor that has priority in the ‘last shot doctrine’ in the courts. Besides, the objective of choosing international law to govern the contract is to provide more comfortably for other parties to enter the contracts, rather than selecting particular domestic law. So, as I recommend you to choose the Malaysia law as governing law, this method is clearly not suitable because it controversy to governing law that you chosen. The object clauses can create legal and practical problems to you in term of quality and specification of the goods you export. Certificate of origin is basic requirement for export goods to other countries, and as discussed before, you needs to obtain a certificate of origin ‘Form D’ from Ministry of International Trade and Industry (MITI) for trade on AFTA, and ‘Form E’ for trade on ACFTA in order to fulfill the CEPT scheme. In other simply meaning, you must to obtain the certificate of origin in order to enjoy the benefit of tariff in the free trade area and simply act as a ‘passport’ that show approval to entering particular market. The packing aspect of goods’ specification creates the problem on the transport of durian to other countries by the strong odour of durian leaking out from the poor packaging. So you need to practice the suitable packaging method for your export durian especially your fresh durian that exported by air shipment. Besides that, you must prepare for the future of sustainable packaging that reflect in the designed in a holistic way and be made from responsibly sourced materials that are safe and effective throughout its lifecycle, meet criteria for performance and cost, meet consumers’ choice and expectations and, finally, it has to be recovered efficiently after use. For the price clauses, you better determine the price that can change over time subject to review and modification because there are fluctuate in the currency exchange among all the different countries that will cause huge lost if there are big differences between the current currency and the currency that agree on the fixed price agreement. Payment clauses also need to be aware because the method of payment will affect your receivable ability, and letter of credit seem more suitable for you because it emphasis more on the seller side through the process that provide more insure on receiving of payment for seller side. Penalty for late payment in this clauses will not only provide extra insure to you through the charges gain for the late payment, but it also help in your financial arrangement due to the on-time payment and assurance of creditability of the buyer through the slightly higher of penalty being set. Delivery and shipment clauses will also raise problem through period time that involve in transport the perishable durian product. So, in order to maintained the product freshness especially when transport at long distance like to China, the date and also time must specify in detail referred to the time of harvest and the available of transportation to prevent any extra days or hours it incurred to transport the product. Besides that, port of shipment is also a critical element in this clause because the distance between the choosing port and the distribution centre determine the product freshness also. For example in China, you can choose the port of Guangzhou because it is considering being a centre for exporting Malaysian durian to China. As I suggest you to choose Malaysian law as the governing law, you need to state this clearly in the clause of governing law. Besides, after state of the governing law is Malaysian law, follow by the jurisdiction will state Malaysia court is the place to resolve dispute. If this never state in the contract, it will depend on court to decide which law apply. The clause of passing of title and risk is also a vital term to consider when there are accidents happen to the goods on the carrier stage or incident of unpaid seller. ? Bibliography 1. AB Teoh. 2008. Exporting and International Trade [access on 15 July 2010] 2. Essential international trade law by Michelle Sanson. 2002 by Cavendish Publishing (Australia) Pty Limited. Available www. cavendishpublish. com. [access on 15 July 2010] How to cite International Trade Law, Papers

Implications Of Labor And The Gig Economy †MyAssignmenthelp.com

Question: Discuss about the Implications Of Labor And The Gig Economy. Answer: The main focus of the reading is the implications of labor and the gig economy, it is a new term which is attracting the attention in the field of news and also in magazines these days. The journal articles are also dealing with this problem. The term gig economy means to understand the two forms of work which incorporates the work via the process of apps and crowdwork. Crowd work is generally referred to the activities based on working which can be completed through the platforms of online (De Stephano 2015; Friedman 2014). These platforms generally deal with many organizations and trying to establish contact with an indefinite number or people through the basis of internet. Work on demand with the help of apps is a type of work which incorporates the traditional activities such as transport, cleaning and other types of clerical jobs are facing challenges through the mode of application which are generally managed by the firms. The paper mainly focuses on the implication which advocates the acknowledgement of the various activities under the umbrella of gig economy in the form of work as the labors risk is eclipsed by the facts such as tasks, gigs or rides (De Stephano 2015; Friedman 2014). It is to be understand that the gig economy is not be treated as a separate economys silo and how it has become the part of a wider phenomena such as escalating of the not standard varieties of employment (Friedman 2014). It also talks about the risks which are linked to these kinds of activities in the context of Rights at work and Fundamental principles which are defined by International Labor Organization which further elaborates the misclassification of the status of the employments of the workers which are based on the established agreements of services, practices of the business and the sectors litigation. The relevant trends are also being evaluated which is the emergence of the workers forms of self organization (De Stephano 2015). Next, the proposals such as the creation of the category based on intermediate status between the independent contractor and employment in order to categorize the workers under the umbrella of the gig economy and other proposals of tentative nature is also being put forward such as giving full recognition of the activities in this particular sector such as work fundamental labor rights and the extension of the important rights of the labor to all the working people by not taking into consideration their status of employment and the acknowledgement of the roles of the social workers while trying to avoid the temptations of the deregulations which are based on the haste (De Stephano 2015; Scheiber 2015). In order to promote the protection of labor under the economy of gig, the primary thing has to be a strong advocacy in terms of having jobs. This could be an important step to challenge the risk of the commoditization (Friedman 2014). The gig economy should be treated as a separate silo of the economy in the labor markets, as gig economy is strongly associated in a wider way with the labor market (De Stephano 2015; Sundarajan 2015). Challenges in the gig economy is a quite enormous and protection must be taken for the shrinking workers only if the opportunity is taking place from this economy. References Sundararajan, A., 2015. The gig economyis coming: What will it mean for work.The Guardian,26, p.2015. Scheiber, N., 2015. Growth in the Gig EconomyFuels Work Force Anxieties.The New York Times. De Stefano, V., 2015. The rise of the'just-in-time workforce': On-demand work, crowd work and labour protection in the'gig-economy'. Friedman, G., 2014. Workers without employers: shadow corporations and the rise of the gig economy.Review of Keynesian Economics,2(2), pp.171-188.

Table of Contents Essays (18692 words) - , Term Papers

Table of Contents QUEST Timeline 2 Copy of QUEST Timeline 3 Parent Notification Form 5 QUEST Grading 7 Standard QUEST Style 8 Social Issue Declaration Form 9 Annotated Bibliography 11 Source Check Directions 12 Source Check Sample 13 Essential Question (EQ): First Draft 15 Consultant Agreement Form 17 Experience Plan 18 Experience Hours Log 21 QUEST Interviews 23 QUEST Interview s (Ex amples ) 24 Creating Outlines For QUEST Papers/Presentations 25 Social Issue Research Paper 26 Social Issue Research Paper (Selected Examples) 29 Social Issue Research Paper Rubric 31 Social Issue Research Presentation 32 Social Issue Research Presentation Rubric 35 QUEST Service 36 Service Plan 39 Service Verification 43 Essential Question (EQ): Updated Draft 45 Policy Paper 47 Policy Paper Rubric 50 Policy Presentation 51 Policy Presentation Rubric 53 Essential Question: Answer Paper 54 Government Advocacy Letter 55 Government Advocacy Letter (Example) 56 QUEST Binder Check (Checklist) 57 QUEST Testimony 59 QUEST Testimony Rubric 61 Ms. McCauley's Guide to Citing Anything! 62 QUEST 2016-2017 Timeline First Semester Ends 1/22 Semester 1 Sept 20 th Quest Kick-Off (Norse Hall) Sept 27 th Social Issue Declaration Form (Eng/Gov) Parent Notification Form (Gov) Oct 11 th Source Check #1 (Eng) Oct 18 th Essential Question First Draft ( Eng/ Gov) Nov 1 st Consultant Agreement Form (Gov) Experience Plan (Gov) Nov 8 th Source Check #2 (1 Ac. Journal) (Gov) Nov 15 th EXPERIENCE Interview Notes (Eng) Nov 29 th Source Check #3 (1 Ac. Journal) (Eng) Dec 6 th Social Issue Research Paper Outline (Eng) Dec 13 th Social Issue Research Paper (Eng) Source Check #4 (Include In Ann Bib) (Gov) Annotated Bibliography #1 (Gov) Jan 9 th to 27 th Social Issue Research Presentation (Gov) Jan 10 th 5 Experience Hours Log (Gov) Jan 17 th Service Plan (Econ) Semester 2 Feb 7 th SERVICE Interview Notes (Eng) EQ Draft (Gov/Eng) Feb 14 th Source Check #5 (1 Ac. Journal) (Econ) Thank You Email (Eng) Feb 28 th Source Check #6 (Eng) March 7th Policy Paper Outline (Econ) March 14 th Policy Paper (Econ) Annotated Bibliography #2 (Eng) March 28 th Service Verification (Econ) March 27 th to April 13 th Policy/Service Presentation (Eng) April 11 th Answer Paper (Eng) Government Advocacy Letter (Econ) April 25 th Binder Check (Eng) April 28 th Last Day to Turn in Work! May 24 th and 25 th Testimonies (Grade in Eng) QUEST 2016-2017 Timeline First Semester Ends 1/22 Semester 1 Sept 20 th Quest Kick-Off (Norse Hall) Sept 27 th Social Issue Declaration Form (Eng/Gov) Parent Notification Form (Gov) Oct 11 th Source Check #1 (Eng /Gov ) Oct 18 th Essential Question First Draft (Gov) Nov 1 st Consultant Agreement Form (Gov) Experience Plan (Gov) Nov 8 th Source Check #2 (1 Ac. Journal) (Gov) Nov 15 th EXPERIENCE Interview Notes (Eng) Nov 29 th Source Check #3 (1 Ac. Journal) (Eng) Dec 6 th Social Issue Research Paper Outline (Eng) Dec 13 th Social Issue Research Paper (Eng) Source Check #4 (Include In Ann Bib) (Gov) Annotated Bibliography #1 (Gov) Jan 9 th to 27 th Social Issue Research Presentation (Gov) Jan 10 th 5 Experience Hours Log (Gov) Jan 17 th Service Plan (Econ) Semester 2 Feb 7 th SERVICE Interview Notes (Eng) EQ Draft (Gov/Eng) Feb 14 th Source Check #5 (1 Ac. Journal) (Econ) Thank You Email (Eng) Feb 28 th Source Check #6 (Eng) March 7th Policy Paper Outline (Econ) March 14 th Policy Paper (Econ) Annotated Bibliography #2 (Eng) March 28 th Service Verification (Econ) March 27 th to April 13 th Policy/Service Presentation (Eng) April 11 th Answer Paper (Eng) Government Advocacy Letter (Econ) April 25 th Binder Check (Eng) April 28 th Last Day to Turn in Work! May 24 th and 25 th Testimonies (Grade in Eng) Parental Notification of Student Participation in QUEST (Due 9/27/2016 in Government) I am the parent or legal guardian of ______________________________________________, who is a Senior at Irvington High School. I understand that as part of the senior curriculum at Irvington High School, he/she will participate in the QUEST project and that his/her successful completion of this benchmark is necessary to graduate from Irvington High School. I understand that

Biography of Henry Clinton, British General

Biography of Henry Clinton, British General Henry Clinton (April 16, 1730–Dec. 23, 1795) was the Commander of the British North American forces during the American War for Independence. Fast Facts: Henry Clinton Known For: Commander of the British North American forces during the American War for IndependenceBorn: About 1730 in Newfoundland, Canada or Stourton Parva, England.Parents: Admiral George Clinton (1686–1761) and Ann Carle (1696–1767).Died: December 23, 1795 in GibraltarEducation: In New York colony and possibly studied under Samuel SeaburyPublished Works: The American Rebellion: Sir Henry Clintons Narrative of His Campaigns, 1775–1782Spouse: Harriet Carter (m. 1767–1772)Children: Frederick (1767–1774), Augusta Clinton Dawkins (1768–1852), William Henry (1769–1846), Henry (1771–1829), and Harriet (1772) Early Life Henry Clinton was likely born in 1730 to Admiral George Clinton (1686–1761), at the time the Governor of Newfoundland and Labrador, and his wife Ann Carle (1696–1767). References are that available post his birth date as 1730 or 1738; English peerage records state the date as April 16, 1730, but list his birth location as Newfoundland and George Clinton did not arrive until 1731. Henry Clinton had at least two sisters who survived to adulthood,  Lucy Mary Clinton Roddam, 1729–1750, and Mary Clinton Willes (1742–1813), and Lucy Mary was born in Stourton Parva, Lincolnshire, England.   Little more than that is known about his childhood: what there is comes primarily from 19th-century brief biographical records and the letters and documents left by Clinton himself. When George Clinton was appointed governor of New York in 1743, the family moved there and it is assumed that Henry was educated in the colony and may have studied under Samuel Seabury (1729–1796), the first American Episcopal bishop. Early Military Career Beginning his military career with the local militia in 1745, Clinton obtained a captains commission the following year and served in the garrison at the recently captured fortress of Louisbourg on Cape Breton Island.  Three years later, he traveled back to England with hopes to secure another commission in the British Army. Purchasing a commission as a captain in the Coldstream Guards in 1751, Clinton proved to be a gifted officer. Swiftly moving through the ranks by buying higher commissions, Clinton also benefited from family connections to the Dukes of Newcastle. In 1756, this ambition, along with assistance from his father, saw him gain an appointment to serve as aide-de-camp to Sir John Ligonier. Seven Years War By 1758, Clinton had reached the rank of lieutenant colonel in the 1st Foot Guards (Grenadier Guards). Ordered to Germany during the Seven Years War, he saw action at the Battles of Villinghausen (1761) and  Wilhelmsthal (1762).  Distinguishing himself, Clinton was promoted to colonel effective June 24, 1762, and appointed an aide-de-camp to the armys commander, Duke Ferdinand of Brunswick. While serving in Ferdinands camp, he developed a number of acquaintances including future adversaries Charles Lee and William Alexander (Lord Stirling). Later that summer both Ferdinand and Clinton were wounded during the defeat at Nauheim. Recovering, he returned to Britain following the capture of Cassel that November.   With the end of the war in 1763, Clinton found himself head of his family as his father had died two years earlier. Remaining in the army, he endeavored to resolve his fathers affairs- which included collecting an unpaid salary, selling land in the colonies, and clearing a large number of debts. In 1766, Clinton received command of the 12th Regiment of Foot.   In 1767 he married Harriet Carter, the daughter of a wealthy landowner. Settling in Surrey, the couple would have five children (Frederick (1767–1774), Augusta Clinton Dawkins (1768–1852), William Henry (1769–1846), Henry (1771–1829), and Harriet (1772).  On May 25, 1772, Clinton was promoted to major general, and two months later he used family influence to gain a seat in Parliament. These advancements were tempered in August when Harriet died a week after giving birth to their fifth child. After she died, Henrys in-laws moved into his house to raise the children. He apparently acquired a mistress at a later point in his life and had a family with her, but their existence is merely mentioned in Clintons surviving correspondence. The American Revolution Begins Crushed by the loss of wife, Clinton failed to take his seat in Parliament and instead traveled to the Balkans to study the Russian army in 1774. While there, he also viewed several of the battlefields from the Russo-Turkish War (1768–1774). Returning from the trip, he took his seat in September 1774. With the American Revolution looming in 1775, Clinton was dispatched to Boston aboard HMS Cerberus with Major Generals William Howe and John Burgoyne to provide assistance to Lieutenant General Thomas Gage. Arriving in May, he learned that fighting had begun and that Boston had fallen under siege.  Assessing the situation, Clinton brusquely suggested manning Dorchester Heights but was refused by Gage. Though this request was denied, Gage did make plans for occupying other high ground outside of the city, including Bunker Hill. Failure in the South On June 17, 1775, Clinton took part in the bloody British victory at the Battle of Bunker Hill. Initially tasked with providing reserves to Howe, he later crossed to Charlestown and worked to rally the dispirited British troops. In October, Howe replaced Gage as commander of British troops in America and Clinton was appointed as his second-in-command with the temporary rank of lieutenant general. The following spring, Howe dispatched Clinton south to assess military opportunities in the Carolinas. While he was away, American troops emplaced guns on Dorchester Heights in Boston, which compelled Howe to evacuate the city. After some delays, Clinton met a fleet under Commodore Sir Peter Parker, and the two resolved to attack Charleston, South Carolina. Landing Clintons troops on Long Island, near Charleston, Parker hoped the infantry could aid in defeating the coastal defenses while he attacked from the sea. Moving forward on June 28, 1776, Clintons men were unable to render assistance as they were halted by swamps and deep channels. Parkers naval attack was repulsed with heavy casualties and both he and Clinton withdrew. Sailing north, they joined Howes main army for the assault on New York. Crossing to Long Island from the camp on Staten Island, Clinton surveyed the American positions in the area and devised the British plans for the upcoming battle. Success in New York Utilizing Clintons ideas, which called for a strike through the Guan Heights via Jamaica Pass, Howe flanked the Americans and led the army to victory at the Battle of Long Island in August 1776. For his contributions, he was formally promoted to lieutenant general and made a Knight of the Order of Bath. As tensions between Howe and Clinton increased due to the latters constant criticism, the former dispatched his subordinate with 6,000 men to capture Newport, Rhode Island in December 1776. Accomplishing this, Clinton requested leave and returned to England in spring 1777. While in London, he lobbied to command a force that would attack south from Canada that summer but was denied in favor of Burgoyne. Returning to New York in June 1777, Clinton was left in command of the city while Howe sailed south to capture Philadelphia. Possessing a garrison of only 7,000 men, Clinton feared attack from General George Washington while Howe was away. This situation was made worse by calls for help from Burgoynes army, which was advancing south from Lake Champlain. Unable to move north in force, Clinton promised to take action to aid Burgoyne. In October he successfully attacked American positions in the Hudson Highlands, capturing Forts Clinton and Montgomery, but was unable to prevent Burgoynes eventual surrender at Saratoga. The British defeat led to the Treaty of Alliance (1778) which saw France enter the war in support of the Americans. On March 21, 1778, Clinton replaced Howe as commander-in-chief after the latter resigned in protest over British war policy. In Command Taking command at Philadelphia, with Major General Lord Charles Cornwallis as his second-in-command, Clinton was immediately weakened by the need to detach 5,000 men for service in the Caribbean against the French. Deciding to abandon Philadelphia to focus on holding New York, Clinton led the army into New Jersey in June. Conducting a strategic retreat, he fought a large battle with Washington at Monmouth on June 28 which resulted in a draw. Safely reaching New York, Clinton began drawing up plans for shifting the focus of the war to the South where he believed Loyalist support would be greater. Dispatching a force late that year, his men succeeded in capturing Savannah, Georgia. After waiting for much of 1779 for reinforcements, Clinton was finally able to move against Charleston in early 1780. Sailing south with 8,700 men and fleet led by Vice Admiral Mariot Arbuthnot, Clinton laid siege to the city on March 29. After a prolonged struggle, the city fell on May 12 and over 5,000 Americans were captured. Though he wished to lead the Southern Campaign in person, Clinton was forced to turn over command to Cornwallis after learning of a French fleet approaching New York. Returning to the city, Clinton attempted to oversee Cornwallis campaign from afar. Rivals who did not care for each other, Clinton and Cornwallis relationship continued to be strained. As time passed, Cornwallis began to operate with increasing independence from his far-away superior. Hemmed in by Washingtons army, Clinton limited his activities to defending New York and launching nuisance raids in the region. In 1781, with Cornwallis under siege at Yorktown, Clinton attempted to organize a relief force. Unfortunately, by the time he departed, Cornwallis had already surrendered to Washington. As a result of Cornwallis defeat, Clinton was replaced by Sir Guy Carleton in March 1782. Death Officially turning command over to Carleton in May, Clinton was made the scapegoat for the British defeat in America. Returning to England, he wrote his memoirs in an attempt to cleanse his reputation and resumed his seat in Parliament until 1784. Re-elected to Parliament in 1790, with assistance from Newcastle, Clinton was promoted to general three years later. The following year he was appointed Governor of Gibraltar, but died in Gibraltar on Dec. 23, 1795, before taking over the post.

These Are the Most Diverse Colleges in America

These Are the Most Diverse Colleges in America SAT / ACT Prep Online Guides and Tips Going to a diverse college offers many advantages. At diverse colleges, you’ll be exposed to a wide variety of people and be given an opportunity to learn from people who are different from you. If you know you want to go to a diverse college, how do you find diverse colleges? Which are the most diverse colleges in the United States? In this article, I will provide you with a list of the most diverse colleges. Furthermore, I'll explain what makes a college diverse, the benefits of going to a diverse college, and how to determine if a specific college is diverse. What Makes a College Diverse? Literally, diverse means showing a great deal of variety. Usually, when people reference diversity at a college, they're referring to the racial and ethnic diversity of the student body. A diverse college will have a significant percentage of students from multiple racial and ethnic groups. However, racial diversity is not the only variable that determines whether or not a college is diverse. Here are other factors that contribute to the diversity of a college: Geographic diversity- Diverse colleges have a higher percentage of out-of-state and international students. Male-female diversity- Schools with more gender balance are more diverse. Faculty diversity- Diverse colleges have more racial and ethnic diversity in their faculties, and their faculties have more gender balance. Economic diversity- A diverse college will have a significant percentage of students from all income levels. There are some other factors that contribute to diversity, but there are fewer available statistics to definitively determine diversity in these areas. The political diversity of a college refers to the percentage of students from different political persuasions. A politically diverse college will have a significant number of conservative, liberal, socialist, and libertarian students. A religiously diverse college will have a large percentage of students from different religious backgrounds; it could have a substantial representation of Catholic, Protestant, Mormon, Jewish, Muslim, and Hindu students. Finally, the percentage of LGBT students contributes to the diversity of a college. A diverse college will have a visible LGBT community and students that openly express different sexual orientations and gender identities. What Are the Benefits of a Diverse College? There are numerous benefits of attending a diverse college. In college, you learn from your peers and fellow students. If you're exposed to more people from different backgrounds, you're likely to gain a better understanding of different types of people and their views. As our economy becomes more globalized, being knowledgeable about various cultures can benefit you in your professional life. Also, if you're a member of an underrepresented group, going to a diverse college can make you feel more comfortable, especially if there's a significant number of students at the college who share your background or beliefs. Similarly, if you come from a diverse high school or neighborhood, you may feel more at home at a diverse college. Furthermore, colleges with diverse faculties may offer a more well-balanced education. Course offerings and instruction at a college can be reflective of the backgrounds and views of the faculty, especially in the humanities and social sciences. Additionally, colleges with diverse faculties show a commitment to diversity and are likely to embrace diversity in their student bodies as well. The List of the Most Diverse Colleges I've given you a list of the top 50 most diverse colleges in the country. Hopefully, you can find at least a few that interest you if you want to attend a diverse college. Niche Niche is a website that provides reviews, rankings, and statistics about neighborhoods and schools. It provides many different college ranking lists from the overall best colleges to the top party schools to the most diverse colleges. The Niche rankings for the most diverse colleges are based on clear criteria that give a fairly accurate measurement of a college's level of diversity. Here's how Niche measures diversity: 20% of the rankings is based on the percentage of international students 20% is based on the percentage of the most represented ethnicity (a lower percentage=more diverse) 20% is based on students' survey responses about the quality of diversity at their colleges 15% is based on the percentage of out-of-state students 10% is based on the percentage of the faculty's most represented ethnicity 5% is based on the ratio of male to female faculty 5% is based on the percentage of students belonging to the most represented income bracket (a lower percentage=more diverse) 5% is based on the ratio of male to female undergraduates The List Many selective private colleges are in the top 50 most diverse colleges, including Pomona, Amherst, MIT, Stanford, Swarthmore, Yale, and Harvard. Interestingly, the entire top 25 is composed of private colleges. Possibly, the reason for this is because private colleges emphasize diversity more in their recruiting and admissions processes. Also, many public colleges have a much higher percentage of in-state students due to lower tuition costs for in-state students and targeted efforts to enroll more in-state residents. Some public universities in the top 50 include California State University-East Bay, Rutgers University-Newark, University of Hawaii at Hilo, and San Francisco State University. These diverse public colleges are located in diverse areas. San Francisco State University School Location Acceptance Rate 1. Pomona College Claremont, CA 14% 2. Amherst College Amherst Town, MA 14% 3. Massachusetts Institute of Technology Cambridge, MA 8% 4. Soka University of America Aliso Viejo, CA 43% 5. Hawaii Pacific University Honolulu Township, HI 64% 6. Stanford University Stanford, CA 6% 7. Brown University Providence, RI 9% 8. California College of the Arts San Francisco, CA 82% 9. Yale University New Haven, CT 7% 10. Swarthmore College Swarthmore, PA 14% 11. Grinnell College Grinnell, IA 35% 12. Barry University Miami, FL 47% 13. Columbia University New York City, NY 7% 14. Chaminade University of Honolulu Honolulu Township, HI 84% 15. University of Miami Coral Gables, FL 40% 16. Wellesley College Wellesley, MA 29% 17. Rhode Island School of Design Providence, RI 27% 18. New York University New York City, NY 32% 19. University of San Francisco San Francisco, CA 69% 20. California Institute of the Arts Santa Clarita, CA 31% 21. Rice University Houston, TX 17% 22. Emory University Atlanta, GA 26% 23. Nyack College Nyack, NY 97% 24. University of Chicago Chicago, IL 9% 25. Holy Names University Oakland, CA 57% 26. California State University-East Bay Hayward, CA 68% 27. Rutgers University-Newark Newark, NJ 54% 28. University of Hawaii at Hilo Hilo, HI 75% 29. The New School New York City, NY 67% 30. University of Bridgeport Bridgeport, CT 64% 31. Carnegie Mellon University Pittsburgh, PA 25% 32. Washington Adventist University Tacoma Park, MD 45% 33. Agnes Scott College Decatur, GA 67% 34. University of Hawaii at Manoa Honolulu Township, HI 80% 35. Harvard University Cambridge, MA 6% 36. Wesleyan University Middletown, CT 20% 37. Princeton University Princeton, NJ 7% 38. University of Houston Houston, TX 58% 39. University of Pennsylvania Philadelphia, PA 12% 40. Manhattanville College Harrison, NY 77% 41. Santa Fe University of Art and Design Santa Fe, NM 100% 42. San Francisco State University San Francisco, CA 60% 43. Pace University New York City, NY 81% 44. Texas Wesleyan University Fort Worth, TX 46% 45. Nova Southeastern University Fort Lauderdale, FL 57% 46. Earlham College Richmond, IN 64% 47. University of Southern California Los Angeles, CA 20% 48. St. John's University-New York Queens, NY 53% 49. Berklee College of Music Boston, MA 19% 50. Clark University Worcester, MA 70% Massachusetts Institute of Technology Student Reviews Niche also offers student reviews of colleges. Here are some comments written by students of some of the most diverse colleges in the United States. I included comments related to the diversity at the college. Pomona College It gets more and more diverse every year in terms of students of color, international students, and low-income students! And the communities aren't completely separated like they tend to be at other schools- each person is a valued part of this community. Love the student body! It's diverse, inclusive, and just a melting pot of all sorts of identities and personalities, all of which come to create a wonderfully complex community on campus. You'll see a lot of variety- no typical culture here. People might think that Pomona is more laid-back than its peer liberal arts colleges due to its California location, but I don't think it is. It's a little bit more humble/mainstream than preppy New England schools, but students take their academics and their futures very, very seriously. Amherst College I feel as if there is a gap between the different races and ethnicities on campus. They seem to congeal together and don't venture out of their created friend groups based on their homeland. I come from a largely homogenous high school, so coming to Amherst was my first chance to ever meet so many people from different ethnicities and backgrounds. It is NOT just white and preppy. Well, it's pretty preppy. But definitely not predominantly white. Stanford University One of the most diverse schools as far as every aspect goes except for international students. But the school is not just ethnically diverse, but very socioeconomically diverse, thanks to the financial aid. The campus is extremely diverse in all aspects. This is fueled by the large number of international students at the school. However outside of Greek organizations the different groups tend to stick together within their own social circles and very few branch out. University of Miami My school is amazing when it comes to diversifying your life. Here you can meet people of different races, religions, backgrounds; you name it, we've got it. I don't regret coming to Miami at all- I love it here! I was a little hesitant coming to a school with so many wealthy people, while I am not at all. But it really hasn't been an issue at all! The student body is so diverse, I can always find people to talk to. University of Miami How Should You Use The List of the Most Diverse Colleges? If you want to go to a very diverse college, you should research the colleges that interest you on the Niche list to determine if they’re schools you should apply to or attend. There are many factors to consider to determine if a college is a good fit for you including location, selectivity, support services, and the majors offered.Look at the school’s website, and use guidebooks, college finders, search websites, and other ranking lists to help you in the college selection process. If possible, consult with teachers, counselors, parents, current students, and alumni. What Should You Do if You Want to Go to a Diverse College, But a School You're Considering Isn't on the List? Just because a school didn't make the list doesn't necessarily mean that it's not diverse. Many big public state universities didn't rank highly for diversity, but big public state schools often have large numbers of students from all different backgrounds, especially if the school is located in a diverse state. Look at the school's website to see if there are student groups that represent a wide variety of interests or yours in particular. Also, you can consult other sources that evaluate or grade the diversity of a college.If you look up a specific college on Niche, you can find its diversity grade as well as a write-up and student reviews about diversity on campus.If you look up a college on College View, you can find the statistics for the student body racial diversity, total numbers of male and female faculty, and the states and countries represented by its students. What's Next? If you want to check out more ranking lists that show a school's commitment to diversity, you can read about the most LGBTQ-friendly schools. Also, if you're looking to go to an elite school with very accomplished students, investigate the most selective colleges. Finally, as you're navigating the college application and selection process, I highly recommend that you read this post about how to do college research. Want to improve your SAT score by 160 points or your ACT score by 4 points?We've written a guide for each test about the top 5 strategies you must be using to have a shot at improving your score. Download it for free now:

Financing A Healthcare Organization Essay Example | Topics and Well Written Essays - 1250 words

Financing A Healthcare Organization - Essay Example If a firm has sufficient net working capital it is assumed to have enough liquidity (ICFAI Center for Management Research ICMR, 2004). In the operating cycle of the firm current assets are converted into cash to provide funds for the payment of current liabilities. Hence, if the current ratio is higher it means that the short-term liquidity of the firm is also higher. Long-term Solvency Ratio - This is one of the leverage ratios. When the analysis of a firm is extended to the long-term solvency, we come into the category of leverage ratios. The leverage ratios are structural ratios and coverage ratios. Structural ratios are based on the proportions are derived from the relationships between debt servicing commitments and sources of funds for meeting these obligations. This ratio measures the extent to which borrowed funds support the firm’s assets. The denominator in the ratio is total of all assets as indicated in the balance sheet. The type of assets an organization employs in its operations should determine to some extent the sources of funds used to finance them. Management/Expense Ratio - An expense ratio is determined through an annual  calculation,  where  a funds operating expenses  are divided by the average dollar value of its assets under management. Operating expenses are taken out of a funds assets and lower the return to a funds investors (Investopedia). Line-item Budget: Line item budgets are used in private industry for the comparison and budgeting of selected object groups and their previous and future estimated expenditure levels within an organization. The line-item budget should include all income and expense associated with the proposed project. The major advantages of the line-item budget are that is easy to prepare, understand and justify. The big disadvantage is that there is no relationship between the budget request and objectives and priorities, and it is different to transfer

Exploring the Marketing Mix Essay Example | Topics and Well Written Essays - 5000 words

Exploring the Marketing Mix - Essay Example I feel that the consumer perceptions of brands and their attitudes toward strong brands is the core element of this lecture, as it all boils down to figuring out consumer preferences and what consumers actually expect and how they behave (Brassington & Pettitt, 2003). The reason I prefer this part is that it also throws light on the bet practises to win a customer, from a company’s or a manufacturer’s perspective. All the arguments in the lecture lead to the fact that brands affect the buying behaviour of consumers to a great extent. A classic example of this is the choice between diet coke and diet pepsi. As a consumer, I have always preferred diet coke over diet pepsi. But I was amazed to find that various tests have actually proved that the quality of diet pepsi to be better than diet coke. But still, diet coke leads the market as the most preferred drink in the diet colas segment (De Chernatony & McDonald, 1998). When the drinks were tested with two matched sample of consumers, the first ‘blind’ (brands were concealed) group preferred diet pepsi. However, when the same test was carried out in an ‘open’ (brand identities were shown) group, they preferred diet coke (De Chernatony & McDonald, 1998). This example indicates that strong brands are important to both companies and consumers. Though product qualities and prices are given great importance, strong brand overrules all these elements, and can influence the decision making process of the consumers to a much greater extent. Hence distinctive brand identity is the most important element in creating a successful and strong brand (Jobber, 2004). It is evident that a successful brand should win consumer trust and should add value to the companies and positively affect the customer perceptions of the brand. Consumers also gain, as strong brands assure them of the quality of the products and act as a certification. The formula put forth by Doyle (1998) states that a successful brand is a

Kudler Fine Foods Computer Information System Essay Example for Free

Kudler Fine Foods Computer Information System Essay Kudler Fine Foods (KFF) is a California-based provider of a variety of high end foods, both local and from around the world, founded by Kathy Kudler in 1998. Since the opening of the first location, Kudler has opened two more locations in the San Diego area; Del Mar in 2000 and Encinitas in 2003 (About, 2011). As a growing business, Kudler’s needs are constantly changing, especially in terms of the company’s information technology and information systems. This paper will discuss the needs of the company in regards to business operations and accounting, as well as discuss the strengths and weaknesses of the systems currently in place. Based on the current technological opportunities, suggestions will be made to further improve these systems, and evaluate any threats that may affect these systems. Primary Findings Business and Accounting Needs Business Needs As a retail company, Kudler Fine Foods needs to run as smoothly and efficiently as possible. Kathy Kudler budgeted over $50,000 for Smith Systems to select and install the existing finance and accounting system, called Retail Enterprise Management System (REMS). REMS provides an Point-of-Sale module, or POS module, to automate all retails sales made, in detail. This system automatically reports this data to the accounting modules, as well as manages all credit/debit card transactions made in the stores. This helps to eliminate data entry errors by limiting the amount of data requiring manual input by employees (Accounting System Overview, 2011). Accounting Needs REMS also addresses the accounting needs of KFF, by providing applications for the general ledger, accounts payable, bank reconciliation, asset management and accounting modules. Each of these modules is interconnected with the POS, which allows for direct transfer of data between modules (Accounting System Overview, 2011). General Ledger Module. The General Ledger module includes the chart of accounts, and transaction details transferred y the POS system. This data is used to create the company’s budget and financial reporting. Due to the electronic transfer of data from the POS to the general ledger, this module requires minimal manual data entry (Accounting System Overview, 2011). Accounts Payable Module. The AP module allows for the check disbursement portion of the business to be done electronically. This module holds vendor data, tax and freight data, and accepts data from other modules that directly affect purchasing (Accounting System Overview, 2011). Bank Reconciliation Module. Data from the accounts payable, accounts receivable and purchase order modules are compiled automatically by the bank reconciliation module. This data is used in cash flow analysis for financial reporting (Accounting System Overview, 2011). Asset Management and Accounting: There was no module created for this portion of the business. Kudler needs to develop an efficient and economical way to track inventory and other assets, other than relying on the leasing company and spreadsheets (Accounting System Overview, 2011). Strengths and Weaknesses in the Current System Strengths The strength in the existing information system is that the modules that are installed reduce the manual data entry, thus eliminating unnecessary errors to the information, by allowing the modules to share information provided by the point-of sale machines. The POS system itself is a major benefit to the IT system as a whole, due to the number of transactions it is able to detail, catalog and parse to the interconnected modules. Secondly, the network set up at each location supports 3-4 POS terminals, an inventory terminal and a server terminal. Each individual network, for each store is providing enough storage power for the needs of each location (Information Technology, 2011). Weaknesses As previously discussed, the network setup has benefits on an individual store basis. However, the system as a whole is not sufficient in running a cohesive, multi location business. Kudler’s inventory systems are completely separate from one another, which could cause a serious issue in asset management within the company. A second weakness is the lack of policy regarding security within the system. This could lead to vulnerability to an internet attack for customer personal information and identity theft. Also, without back up procedures, there is no way for Kudler to prevent data loss, should the network(s) fail. Recommendations Based on the information above, the recommendation for Kudler and KFF would be to first, establish a set of security policies and back up procedures, in order to prevent hacking and data loss, respectively. In order to do so, Kudler would best benefit by reestablishing a working relationship with Smith Systems. Smith would then set up and maintain an offsite server that will service all three KFF locations, and allow the company to consolidate the three separate inventory systems. The offsite server would then serve as a repository for all accounting, inventory and human resources related items. Doing so will cut down the potential staffing and duplication in the inventory system. Additionally this would reduce the number of servers required, and thus reduce the potential of IT related hardware issues. Conclusion To conclude, Kudler Fine Foods uses and information system established by Smith Systems that sufficiently handles point-of-service retail sales receipts, and disburses the information to the appropriate accounting software modules. The modules receive the financial data from the sales to create financial reports, budgeting reports, and analyze accounts receivable and accounts payable. The system also maintains ordering and purchase order data, however, it does not maintain a single inventory module, which could track the sales of these items after the goods are received in the individual stores. In order for this to occur, Kudler needs to establish a solid set of security measures and procedures in the case of data loss. To address this, Smith Systems can be contracted to maintain an offsite server with security maintenance. This will help to prevent internet attacks by hackers searching for customer identification and credit card information. Once these changes are in effect KFF should see an increase in efficiency in the current systems in use.

Java Programming Language Essay -- Computers Technology Programming La

Java: It's not just for breakfast anymore! The World-Wide Web as it is today reminds me of a bad date I had once; boring, flat, and unexciting. It does absolutely nothing for me. The pages are limited by the specifications of HTML which calls for a two-dimensional layout and a static page. I for one am looking for some new element. A new angle if you will. Something to jolt some life into The Web. Sun Microsystem's Java will bring a new interactive element to the Web. It is designed to enhance the browsing experience and take us into the next generation of The Web. "Java is an object-oriented language that adds animation and real-time interaction through in-line applications (called applets)." (Network Computing) So you are asking yourself, What is Java? How does it work? "Java is a simple, object-oriented, multithreaded, garbage-collected, secure, robust, architectural-neutral, portable, high-performance, dynamic language. The language is similar to C and C++ but much simpler. I think Eric Schmidt, Sun's Chief Technical Officer, put it best when he said, "Java is C++ without the guns, knives, and mace. It was designed for a consumer devices market, to allow applications to move among the devices in a secure manner." (P.C. LETTER) Java programs are compiled into a binary format that can be executed on many platforms without recompilation." (Dr. Dobb) It works by converting the code into a format the Java interpreter can understand. The code or "applets" can be embedded in any standard HTML page. An applet can do most anything a regular C program can do. It is equally complex as C for any given task. For the technically impaired, Java is a highly flexible product for developing programs for The Web. It is capable of carryi... ...5, v20 n8 p56-62 "Java and Internet programming: similar to C and C++ but much simpler" van Hoff, Arthur INFOWORLD; May 1995, v17 n22 p16 "Netscape inks pact with Sun, Macromedia" Wingfield, Nick LAN TIMES; June 1995, v12 n12 p44 "Sun, Netscape to wake up Web users" Raynovich, R. Scott MacUser; Sept 1995, v11 n9 P31 "The Internet becomes multimedia-savvy:Macromedia, Sun nab Netscape Navigator" Snell, Jason Network Computing; August 1995, v6 n9 p48-49 "Next generation Web Browsing" Kohlhepp, Robert Newsbytes; July 1995, pnew07180011 "Sun Microsystems Intros First Java Application" Bowers, Richard P.C. Letter; June 1995, v11 n9-10 p5 "Sun's Schmidt explains Java strategy" Author not given PCWEEK; June 1995, v12 n22 p14 "Sun's Java technology perks up WWW; Java language and HotJava browser provide extensiblity to the World-Wide Web" Sullivan, Eamonn

Titration Journal

E r J. Biochem. 40,177-185 (1973) u. Intracellular Titration of Cyclic AMP Bound to Receptor Proteins and Correlation with Cyclic-AMP Levels in the Surviving Rat Diaphragm Lien DO KHAC,Simone HARBON Hubert J. CLAUSER and lnstitut de Biochimie, Universit6 de Paris-Sud, Orsay (Received April 9/July 17, 1973) Extracts prepared from rat diaphragms incubated with or without theophylline and/or epinephrine have been tested for their total cyclic AMP content and for their ability to bind exogenously added cyclic [â€Å"]AMP.Less cyclic [3H]AMP can be bound inthe extracts after theophylline and/or epinephrine treatment indicating that the rise in cyclic AMP level was accompanied by a n increase in the quantity of cyclic AMP bound intracellularly to the cyclic AMP-dependent protein kinases. Maximum cyclic AMP binding capacities, as measured by total cyclic AMP exchanges, were however identical in all cases. Accurate estimations of intracellular binding of cyclic AMP have been correlated with the level of cyclic AMP in the tissue : the reaction seems to obey simple saturation kinetics, a n apparent intracellular K d for cyclic AMP has been evaluated as 330 nM.The findings are consistent either with a real difference in the intracellular binding constant as compared to that measured in vitro (28 nM) or with the fact that the cyclic nucleotide in the cell may not all be available for the kinase protein receptors. They also suggest that the method described may prove useful for studying any possible intracellular control beyond the step of cyclic AMP synthesis.Regulation of cellular metabolism by adenosine 3†² :5†²-monophosphate (cyclic AMP) [I], its mediation through complex protein kinases [2,3] and the mechanism of the activation of these enzymes [4–61 have been well documented within the past years in the eukaryotic cell. Activation has been demonstrated to occur according to Equation (1) through a n interaction of cyclic AMP with the regulatory subuni t (R) of the enzyme, leading to a dissociation of this subunit from the catalytic subunit (C) which is thus activated. RC cyclic AMP + R cyclic AMP C . (1) + +However completely satisfactory correlations between the levels of intracellular cyclic AMP and its ultimate metabolic effects have been in many cases difficult to obtain. Striking examples for this situation are to be found in the results of Craig et al. [7] in rat diaphragm, of Stull and Mayer [8] in rabbit skeletal muscle concerning the regulation of phosphorylase activation, of Schaeffer et al. [9] and Miller et al. [lo] concerning regulation of glycogen metabolism in adrenalectomized rats, and of Harbon and Clauser [Ill This work is dedicated to Professor E. Lederer for his 65 th anniversary. Abbreviations.Cyclic AMP; adenosine 3†²: 5†²-monophosphate. in the rat uterus stimulated by prostaglandin El or E,. I n all these cases, cyclic AMP levels may be elevated without eliciting the expected metabolic responses. Two hypotheses have been formulated to explain these obvious discrepancies, either a decrease in the activation of the enzymes mediating cyclic AMP action within the cell, or a compartmentalization of the intracellular nucleotide. Hence it seems necessary to measure directly the degree to which the first step of the activation sequence (Equation 1)reflects the apparent intracellular cyclic AMP concentrations.This might be achieved by establishing in intact cells or tissues, correlations between the levels of intracellular cyclic AMP under welldefined physiological conditions, the extent to which it is bound to the specific receptor protein and the extent to which the complex protein kinases are in the active state. Satisfactory correlations between cyclic AMP levels and protein kinase activation have been recently established in various tissues by Corbin et al. [I21 and Soderling et al. [13].The present work was to investigate if correlations could also be obtained between intracell ular cyclic AMP levels and the amounts of intracellular cyclic AMP bound to receptor protein (R cyclic AMP) in the surviving rat diaphragm incubated with or without theophylline and epinephrine. The results reported demonstrate that – E r J. Biochem. 40 (1973) u. 178 Intracellular Titration of Cyclic AMP-Receptor Protein Binding precise titrations of endogenous cyclic AMP bound versus cyclic AMP present in the intact tissue may be obtained.An apparent Kd value for the intracehlar cyclic AMP binding is observed which differs widely from the K d of the same binding established in vitro [14-161. This method may prove to be useful for studying the modification of cyclic AMP binding under conditions where the formation and breakdown of cyclic AMP does not seem to be affected. A preliminary report of these results has been presented [17]. MATERIALS AND METHODS Cylic AMP was obtained from P L Biochemicals Inc. , theophylline and Tris from Merck (Darmstadt), Na,ATP 4 H,O, L-epinephri ne bitartrate from Calbiochem.Cellulose ester membrane filters (HA 0. 45 pm, 24 mm) were purchased from Millipore Corp. All reagents used were products of Prolabo (reagent grade). Cyclic [3H]AMP was a product of New England Nuclear Inc. , specific activity 24 Ci/ mmol. Animals were Wistar rats weighing about 200 to 300 g and fasted 24 h before the experiments. Tissue homogenizations were performed with an Ultra Turrax homogenizer. – The reaction mixture for the binding assay contained in a final volume of 250 p1, 20 mM TrisHC1 buffer pH 7. 5, 10 mM MgCI,, 6. 7 mM theophylline and cyclic [3H]AMP a t various concentrations as indicated.The reaction was initiated by the addition of a n aliquot of diaphragm extracts equivalent to 70- 150 pg protein. Method B. I n this case, cyclic [3H]AMPwas added to the homogenizing medium a t saturating concentrations up to 0. 2 p M a t 0 â€Å"C, centrifugation was carried out immediately and cyclic [3H]AMP bound measured directly on the extr act. Cyclic [3H]AMP bound to the proteins, under either condition, was determined after different incubation times at 0 â€Å"C: the reaction mixtures were then diluted to 3 m l with cold buffer (20mM TrisHC1, 10mM MgCl,, pH 7. 5) and passed through cellulose acetate Millipore filters (0. 45 pm).The filters were washed with 25ml of the same buffer, dried and counted in i 0 ml scintillation fluid, in a Packard Tri-Carb liquid scintillation spectrometer. Results were expressed as pmol cyclic AMP bound/mg protein ; the concentration of endogenous unlabelled cyclic AMP has been always taken into account for the estimation of the specific activity of cyclic [3H]AMP present in the incubation medium. Incubation Procedures The animals were killed by decapitation. The diaphragms were rapidly removed, freed from connective tissue, cut to small pieces, pooled and divided into equal parts. 200-250 mg tissue were preincubated in 2. ml Krebs-Ringer-bicarbonate buffer pH 7. 4, gas phase (95O/, O, , 5O//, CO,) for 30 min a t 37 â€Å"C, in the absence or presence of 10 mM theophylline. Incubations were then performed in the absence or presence of epinephrine (5 pM) for varying periods of time. Extraction of the Tissue Standard Binding Assays for Cyclic A M P Two methods have been deviced to extract the tissue and estimate the binding of exogenous cyclic [3H]AMP to the extracted proteins, both slightly modified from the method defined by Walton and Garren [15]. Method A . The tissue was homogenized a t 0 â€Å"C in 3 ml of one of the following solutions: 20 mM TrisHCl buffer pH 7. or 20 mM sodium acetate pH 7. 5 or 4 mM EDTA pH 6. 0. Theophylline (10 mM) was always present in the various homogenizing media in order to minimize any degradation of cyclio AMP by phosphodiesterase present in diaphragm extracts. A first centrifugation was carried out for 5 min a t 3000 x g , followed by a second one a t 50000 x g for 30min. The supernatants will be referred to as Tris extract, ac etate extract and EDTA extract. Assay for Cyclic-AMP Levels For cyclic AMP assay, the tissue was homogenized in 3 ml cold 7 trichloroacetic acid and centrifuged for 30 min a t 50000 xg.After addition of 0. 1 ml N HC1, the supernatants were extracted 7-8 times with twice their volume of cold ether and evaporated to dryness. Total levels of cyclic AMP in the tissue trichloroacetic acid extract were determined according to Gilman using a protein b a s e and the heatstable inhibitor prepared from rabbit skeletal muscle [161. I n some instances, cyclic AMP content was also evaluated in the Tris and acetate extracts. Proteins were precipitated by trichloroacetic acid and extracts processed as described above. Proteins in the extracts were determined according to Lowry et al. 18] using bovine serum albumin as a standard. RESULTS AXD DISCUSSION Total Cyclic-AMP Levels in Rat Diaphragm. Effects of Epinephrine and Theophylline In order to study the cyclic AMP binding capacity of rat diaphragm proteins and its possible rnodification under the influence of epinephrine, it seemed necessary to test the first effect of the catecholamine, viz. the rise in the tissue cyclic AMP level under our experimental conditions. Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 1. Total cyclic A H P levels in trichloroacetic acid extracts of rat diaphragm.Effect of epinephrine and theophylline R a t diaphragms (200-250 mg) were preincubated for 30 rnin a t 37 â€Å"C in 2. 5 ml Krebs-Ringer-bicarbonate buffer (0, 95 °/0-C0, 50/0) in the absence or presence of 10mM theophylline, Incubation was then performed for 5 rnin with or without 5 pM epinephrine. The tissue was then homogenized in 7O/, trichloroacetic acid for cyclic AMP assay as described under Methods. Levels of cyclic AMP were expressed as pmol cyclic AMP/100mg wet tissue and as pmol cyclic AMP/mg soluble protein (as estimated by the Lowry procedure in the Tris extract.Values are means f S. E. M. of 5 di fferent experiments Incubation condit,ions Total cyclic AMP TheoDhvlline EDineDhrine pmo1/100 mg pmol/mg wet tissue soluble protein 41 f 8. 0 20. 5 f 4. 7 104 & 1. 1 52 & 0. 47 93 f 4. 5 46 & 2 350 f 21 170 f 10. 7 179 Table 3. Distribution of cyclic [3H]AMP-bindingfractions i n different hom. ogenutes from rat diaplwagms incubated with or without epinephrine Preincubation and incubation conditions as described in Table 2. Tissues were homogenized in 3 ml 20mM TrisHCI, p H 7. 5, 4 mM EDTA or 20 mM sodium acetate pH 7. and centrifuged for 5 rnin at 3000 x g, the supernatants were centrifuged once more at 500OOxg for 30 min yielding extract 1 and pellet 1. The sediment of the first centrifugation was resuspended in 1. 5 ml of the corresponding buffer and centrifuged at 500OOxg for 30 min giving extract 2 and pellet 2. Binding activity for cyclic rSH]AMP was measured in each fraction as described in the text under method A and was expressed as pmol cyclic AMP bound/l00 mg wet tissue Fr action Cyclic AMP bound in EDTA Acetate Tris extract extract, extract, 5 yM noepinoepino epinephrine nephrine nephrine – + + + + – :Lhrine pmo1/100 mg wet tissue Extract 1 Extract 2 Pellet 1 Pellet 2 15. 70 1. 47 0. 76 1. 49 14. 90 1. 54 0. 83 1. 50 15. 30 1. 35 0. 80 1. 10 9. 40 0. 80 0. 44 0. 39 Table 2. Cyclic A M P levels in different extracts obtained from epinephrine-treated and untreated rat diaphragms Preincubation with 10 mM theophylline and incubation conditions in the absence or presence of 5 pM epinephrine as in Table 1. Diaphragms were homogenized in three different solutions: cold 7O/, trichloroacetic acid, Tris-HC1 pH 7. 5 or acetate p H 7. 5 as described under methods.Centrifugation was carried out for 30 rnin at 50000 x g. Soluble Tris extract, acetate extract and their corresponding sediments were deproteinized by 7 o/o trichloroacetic acid before cyclic AMP assay Incubation with epinephrine None 5wM Total cyclic AMP in Trichloroacetic 20 mM acetate a cid extract pellet 57 280 – 20 mM Tris extract pellet 48 218 9. 5 26 extract pellet 45 242 pmo1/100 mg wet tissue – 8. 5 8. 3 As shown in Table 1, epinephrine (5 pM) in the absence of theophylline increases (by a factor of 2. 5) the total cyclic AMP content of rat diaphragm extracted by trichloroacetic acid.Theophylline alone (10 mM) had a stimulating effect, double; when both compounds were used together, the rise in cyclic AMP levels was 8- t o 9-fold, reaching 350pmol cyclic AMP/100 mg wet tissue. When cyclic AMP was assayed in either acetate or Tris extracts after deproteinization with trichloroacetic acid the values obtained were identical t o those found when the diaphragms were directly extracted with trichloroacetic acid ; hence almost none of the cyclic nucleotide in these extracts was associatcd with membrane-bound fractions (Table 2). Eur. J. Biochem. 0 (1973) Location of Cyclic AMP-Binding Fractions Table 3 shows the distribution of cyclic AMP binding activ ity in various fractions of three rat diaphragm homogenates measured by method A : in all cases more than goo/, of this activity was recovered in the 50000 x g supernatant, almost no cyclic AMP binding occurred in the pellets. Preincubation of the diaphragm with epinephrine did not modify the percentage distribution of the radioactive nucleotide between the supernatants and the pellets, hence subsequent experiments have been performed on the soluble extracts.On the other hand, in the case of epinephrine-treated diaphragms, less exogenous labelled cyclic AMP (about 50-60 °/0) was bound to the various fractions, indicating a decrease in the binding capacity of the extract as compared to the untreated diaphragm. Dilution by endogenous cyclic AMP cannot explain the effect of epinephrine, since allowance was made for this parameter (see Methods) ; the phenomenon was consistently reproducible and will be further substantiated and discussed below.The binding capacities of the various ext racts for cyclic E3H]AMP have also been verified in the absence of any free endogenous cyclic AMP after removal of the latter by filtration through Sephadex G 50 (1x 37 cm) columns, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7. 5 a t 4 â€Å"C. I n these experiments, the detail of which w l not be reported in i l the present manuscript, the effect of epinephrine was still observed, when binding was measured on the main protein peak emerging with the void volume of the columns. When the corrections outlined in the 180 Intracellular Titration of Cyclic AMP-Receptor Protein Binding Z A 0. 51 / 0 20 40 60 Time ( m i n ) l / f r e e cyclic AMP (nM-‘) l / f r e e cyclic A M P (nM-‘) Fig. 1. The time wurse and cyclic-AMP-concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method A ) . (A) Diaphragms were incubated for 30 min in the presence of 10 mM theophylline and extracted with Tris HCI buffer (method A). Cyclic AMP binding was estimated in the presence of various concentrations of cyclic E3H]AMP: 20nM ( 0 – 0 ) ; 60nM ( – ) 0 0 ; SO& (A-A); 100 nM ( –) #-. , a t 0 â€Å"C. The react,ion mixtures contained in a final volume of 2. 5 ml, 20 mM Tris-HC1 buffer, pH 7. , 10 mM MgCI,, 6. 5 mM theophylline. The reaction was initiated by the addition of 930 pg protein. At the indicated times, aliquots were pipetted, immediately diluted with cold 30 mM Tris-HC1buffer pH 7. 5,lO mM MgCl, and passed on the Millipore filters. Filters were washed with the same buffer, dried and counted. Binding activity is expressed as pmol cyclic AMP bound/mg protein. (B) Data obtained from similar experiments where binding for cyclic AMP was performed a t 0 â€Å"C, for 1 h, in the presence of cyclic [aHIAMP ranging from 12 nM to 110 &I. Double-reciprocal plot, according to Klotz [25] Fig. 2.Cyclic-AMP-Concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method B ) . Binding assays were carried out as described under method B. Various concentrations of cyclic [3H]AMP ranging from 12nM to 200 nM were added directly to the homogenizing medium for preparing extracts from epinephrine treated (A-A) and untreated (0-0) rat diaphragms. Aliquot,s of the extracts were filtered through Millipore filters, dried and counted. Double-reciprocal plot, according to Klotz [25] present paper were applied to these figures, the results were essentially identical to those obtained with the unfiltered extracts.Specificity. Kinetics and Concentration Dependence of Exogenous Cyclic-AMP Binding in the Extracts Specificity of cyclic AMP binding has been assessed by dilution experiments of cyclic [3H]AMP (100 nM) with unlabelled nucleotides (adenine, AMP, ATP, cyclic AMP) a t molar concentrations equalling up t o 100 times cyclic [3H]AMP concentrations. I n no case, except with unlabelled cyclic AMP, the amount of radioactive material bound to proteins by either method A or B was significantly reduced (the details of these experiments are not reported).When various concentrations of cyclic [3H]AMP were added to diaphragm extracts (after homogenization and centrifugation) and the binding reaction (method A) carried out for different incubation times at 0 â€Å"C (Fig. I), it appears that saturation was obtained at a concentration of 80 nM for the cyclic nucleotide which essentially coincides with previously published data [14-161 and that binding equilibrium was reached a t p H 7. 5 and 0 â€Å"C after less than 60 min incubation. It has also been verified that with the protein concentration used (70-150 pg in 250 pl) binding of cyclic AMP was directly proportional to the amount of added proteins.From a reciprocal plot of cyclic AMP binding versus cyclic AMP concentration (inset of Fig. I), an apparent Kd of 33 nM can be calculated. When similar experiments were performed by adding various concentrations of cyclic [3H]AMP into the homogenizing medium (method B) and using diaphra gms which have been incubated in the presence and absence of epinephrine, the double-reciprocal plots of Fig. 2 were obtained. The apparent Kd values calculated with this method (45 nM) are in the same range as with method A.I n addition this figure shows that epinephrine treatment of the diaphragms does not modify this Kd but decreases the amount of exogenous cyclic AMP which can be bound to the extract proteins. By comparing exogenous cyclic AMP binding values obtained with methods A and B, it appears (Table 4) that when cyclic [3H]AMPwas added to the Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 4. Comparison of exogenous binding of cyclic [SII]AMP to diaphragm extracts by method A or method B. Rat diaphragms were incubated with theophylline in the absence or presancc of 5 p M epinephrine.Extracts in Tris-HC1 were prepared as described under method A for subsequent binding of cyclic [3H]AMP (100 nM), 1 h, a t 0 â€Å"C. A second series of extracts wer e prepared in the same way but in the prescnce of 100 nM cyclic [3H]ABIP in the homogenizing medium (method R); binding of cyclic [3H]AMP was measured in a n aliquot immediately after centrifugation at 0 â€Å"C (about 1 h after the end of incubation). Values are expressed as pmol bound cyclic AMP/mg protein. Numerals within brackets indicate number of experiments Method Cvclic A P bound with M 5 pM epinephrine no epinephrine pmol/mg protein 4 f 0. 22 (9) 4. 80 5 0. 2 (5) 181 6 t e . ;? 4 Q Q E A B 2 f 0. 13 (9) 3 f 0. 19 (5) 0 I I I 30 60 90 * Time (rnin) homogenization medium (extract B) higher binding values were obtained both with epinephrine-treated and untreated diaphragms, than with method A. This demonstrates that some additional binding of endogenous cyclic AMP occurred during the homogenization and fractionation procedures, which tends to decrease the amount of unoccupied binding sites available for exogenous cyclic [3H]AMP. Hence method B has been currently used to measu re exogenous cyclic AMP binding, since the values obtained with this method seem to reflect intracellular conditions more accurately.Fig. 3. Time course of cyclic [3H]AMP binding in extracts from rat diaphragms incubated in the absence or presence of theophylline orland epinephrine. Half rat diaphragms were preincubated in the absence (m, A ) or in the presence ( 0 , 0 ) of 10 m31 theophylline for 30 min at 37 â€Å"C. Epinephrine (5 pM) was added ( A , 0 )and incubation continued for 5min. Tissue was homogenized in 1. 5 ml Tris-HC1 buffer containing 200 nnf cyclic [3H]AMP and centrifuged at 5000xg for 10 min at 0 â€Å"C.Binding of cyclic [3H]AMP was measured in aliquots of the supernatant at the times indicated, through Millipore filtration, t = 0 corresponds to the onset of the extraction. Results are expressed as pmol cyclic AMP bound/ mg protein (without correction for cyclic AMP exchange) Effect of Theophylline and Epinephrine Treatment on the Binding of Exogenous Cyclic [3H ]AMP by Diaphragm Extracts Fig. 3 shows the results of a typical experiment in which diaphragms have been incubated in the absence or presence of theophylline and epinephrine. Homogenization has been performed according to method B, the centrifugation time of the homogenate kept to a inimum (10 min), and the binding capacity for cyclic [3H]AMP determined a t different times. As may have been expected, this cyclic [3H]AMP binding (which measures the residual binding capacities of the extracts) was, in the course of the whole titration period, inversely related t o the amount of endogenous cyclic AMP present in the relevant extracts (see Table 1). Hence the agents which increase the intracellular cyclic AMP level appear to decrease the amount of binding sites available for exogenous cyclic [3H]AMP, probably through an increase of endogenous cyclic AMP binding to the receptors.I n order to titrate endogenous binding of cyclic AMP accurately, experiments were designed to estiEm. J. Bioc hem. 40 (1973) mate the total binding capacities of the extracts through complete exchange of endogenously bound cyclic AMP with cyclic [3H]AMP, and also to estimate the actual amount of exchange occurring in the extracts between endogenous bound unlabelled cyclic AMP and exogenous cyclic [3H]AMP during the titration period. A precise knowledge of these two parameters is required for the determination of the binding sites occupied by endogenous cyclic AMP at the moment where the tissues are homogenized.Cyclic-AM P Exchange and Determination of Maximal Binding Capacities Total cyclic AMP exchange has been measured under the conditions defined by Wilchek et al. [19] for parotid gland and skeletal muscle : extracts from both treated and untreated diaphragms were f i s t incubated at 0 â€Å"C with cyclic [3H]AMP (100 nM) under binding conditions of method A and then allowed t o exchange with 1 pM unlabelled cyclic AMP at 20 â€Å"C in the presence of 100p. M ATP and 10mM MgCl,. Fig. 4 shows that almost complete exchange of the bound labelled nucleotide occurred within 30 min, 182Intracellular Titration of Cyclic AMP-Receptor Protein Binding 0 10 20 30 40 50 60 Time (min) 70 80 90 Fig. 4. Exchange of bound cyclic [SHIAMP. Extracts were prepared from epinephrine-treated ( + o ) and untreated (0-0) rat diaphragms. Binding of cyclic [3H]AMP was carried out a t 0 â€Å"C in a volume of 2. 5 ml with 500 pg proteins, and 100 nM cyclic r3H]AMP in Tris-HC1 buffer, MgCl, and theophylline a t the concentrations described for the standard binding assay. After 1-h incubation, 1 pM unlabelled cyclic AMP and 100 pM ATP were added and the mixture allowed to stand at 20 °C.At the different times indicated in the figure, aliquots corresponding t o 50 pg protein were pipetted, rapidly diluted with 20 mM Tris-HC1 buffer, 2. 5 mM MgC1, p H 7 5 and filtered through Millipore filters. The filters . were washed with the same buffer, dried and counted. Results are expressed as pmol/ mg protein 0 30 60 90 120 Time (rnin) 180 240 – Total binding capacities of the proteins could thus be measured by incubating the extracts first with 100 nM unlabelled cyclic AMP a t 0 â€Å"C and carrying on the exchange reaction in the presence of 1 pM cyclic I13H]AMP at 20 â€Å"C for 1-2 h ; the values obtained averaged 8. -9. 5 pmol cyclic [3H]AMP/mg soluble protein, both with epinephrine-treated and untreated diaphragms. These results were confirmed by direct assay of bound cyclic AMP: the extracts have been fully saturated with unlabelled 1pM cyclic AMP and filtered as described. After washing the Millipore filters, bound cyclic AMP was extracted by cold 7 O/, trichloroacetic acid and the cyclic nucleotide was directly assayed according to Gilman [16]. The average value was 9. 8 f 0. 4 pmol cyclic AMP bound per mg protein, which is of the same order of magnitude as the amount of bound cyclic [3H]AMP calculated above.Previously published data are in close agreement wi th these values. Walton and GarFen [15] reported maximal binding capacities of 9. 8 pmol/mg protein for adrenal extracts, whereas Gilman [l6] found a total binding of 12pmol/mg protein in muscle extracts. The values for maximal cyclic AMP binding are very low as compared t o the total endogenous cyclic AMP present in the extract (46 pmol/mg protein with the theophylline-treated diaphragm and 170 pmol/mg protein with the epinephrine theophylline-treated diaphragm).It must be added that the binding proteins, saturated with cyclic AMP or not, were almost completely retained on the Millipore filters, and that endogenous cyclic AMP, not Fig. 5. T i m e course of cyclic A M P exchange under binding (0 â€Å"C) and exchange (20 â€Å"C} conditions. Extracts were prepared from epinephrine treated (0,A ) and untreated ( 0 , A) r a t diaphragms. Binding of cyclic AMP was performed as described in Fig. 2 in the presence of 100 nM cyclic AMP for 60 min at 0 â€Å"C. A t the end of the bindin g reaction 1 pM cyclic [3H]AMP was added t. the different extracts, in the absence (A, A ) or presence ( 0 , 0 ) of l00p. M ATP. The reaction mixtures were maintained a t 0 â€Å"C for 2 h and then at 20 â€Å"C (arrow) for 2 more hours. At the different times indicated on the figure, aliquots corresponding t o 70 pg protein were pipetted and treated as in Fig. 4. Results are expressed as cyclic rH]AMP bound in pmol/mg protein. bound to these fractions, was quantitatively recovered in the Millipore filtrates after trichloroacetic acid extraction. The extent t o which this â€Å"free† cyclic AMP may or not be bound to other proteins is presently not known.Cyclic-AMP Exchange under Binding Conditions The extent of cyclic AMP exchange under binding conditions (0 â€Å"C, 1 h, 100 nM cyclic AMP) must be controlled if corrections for simultaneous exchange have to be applied t o binding data: extracts of rat diaphragms treated with theophylline and theophylline epinephrine were first saturated with 1 O O n M unlabelled cyclic AMP (binding conditions) and then exchanged with 1 pM cyclic [3H]AMP but a t 0 â€Å"C. After 2 h, the temperature was raised to 20 â€Å"C and completion ofthe exchange measured after 1-2 h further incubation.Fig. 5 shows that a t 0 â€Å"C, within 1h incubation time, which are the conditions described above for the binding assay, about 200/, of total sites were exchangeable. Under these conditions, ATP and Mg ions slightly increase the exchange velocity. I n addition, this figure confirms that a t 20 â€Å"C total exchange capacities were identical for epinephrine-treated and untreated diaphragms ; hence initial + + Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser 183 Table 5. Relationship between intracellular cyclic A M P levels and cyclic AM P binding in extracts from diaphragm incubated under various conditions Diaphragms were incubated with or without 10 mM theophylline for 30 min at 37 â€Å"C, 5 pM epin ephrine was added where indicated and incubation continued for varying times. From each incubation, half a diaphragm was extracted by trichloroacetic acid for cyclic AMP estimation. The other half was homogenized with Tris-HC1buffer lOOnM cyclic [3H]AMP(method B) for exogenous cyclic AMP binding after 1 h a t 0 â€Å"C; maximal binding capacities were determined in the same extracts a t 20 â€Å"C in the presence of 1 pM cyclic [3H]AMP under conditions described for cyclic A P exchange.R. esults are expressed as pmol cyclic AMP/mg M protein. Endogenous binding values were calculated as the difference between maximal binding capacities ( A )and exogenousbinding ( B ) and corrected for the 200/, exchange + Incubation conditions Theophylline 10 mM Epinephrine 5t*M Time Cyclic AMP Total level Maximal binding Exogenous capacity binding (a) (b) Endogenous binding (a-b) corrected min pmol/mg protein – – – + + + + + + 0 2 10 30 5 5 20. 5 52 43 38 46 170 f 4. 7 & 0. 47 f2 f 10. 7 9. 6 f 0. 9 9. 4 f 0. 1 9. 20 9. 40 8. 9 5 0. 73 8. 9 & 0. 85 5. 35 f0. 40 4. 50 f 0. 133 4. 40 4. 70 4. 46 f 0. 20 2. 7 f0. 224 5. 31 6. 13 6 5. 5 5. 53 7. 77 differences in residual binding capacities reflect variations in the degree of saturation of the receptor proteins by endogenous cyclic AMP, rather than modifications of their maximal binding capacity. 1 Titration o Endogenous Cyclic-AMP Binding in Rat f Diaphragm. Effects of Theophylline and Epinephrine Since total binding capacities of the receptor proteins in the extracts and the amount of exogenous cyclic [3H]AMP bound by these extracts after homogenization may be estimated, it appears possible to calculate endogenous cyclic AMP bound in the intact organs, correcting for a 2001, exchange during the titration period.Table 5 summarizes the results of a series of experiments where diaphragms have been incubated under conditions which modify endogenous levels of cyclic AMP :in every case, half of the diaphragm was extracted with cold trichloroacetic acid (see Methods) for the assay of intracellular cyclic AMP levels: the second half was extracted according to method B for the estimation of exogenous cyclic [3H]AMP binding and of total cyclic AMP binding capacities. The endogenous cyclic AMP bound was calculated from the latter experimental data.This table definitely establishes that the average values obtained for the intracellular binding of endogenous cyclic AMP in the intact organ seem to correlate with its cyclic AMP levels. A reciprocal plot of intracellular binding versus intracellular cyclic AMP concentrations (Fig. 6) shows that this correlation fits simple saturation kinetics very accurately. I n the unstimulated diaphragm (no theophylline nor epinephrine added to the incubation medium) about 50 °/, of the available binding sites are occupied by endogenous cyclic AMP; this Eur. J. Biochem. 40 (1973) -0. 002 I 0. 002 l/Free cyclic AMP (nM-‘) 0 0. 004 . Fig. 6. Reciprocal plot of intracellular cyclic A M P levels and cyclic A M P binding in rat-diaphragm extracts. Data arc obtained from experiments performed as described in Table 5 and replotted according t o the Klotz equation. The intercept on the y axis yields a n estimate of the number of binding sites and the x intercept provides a n estimation of the intracellular apparent dissociation constant. Statistical analysis of the data were performed according to Cleland [26] using a Wang electronic calculator alue increases to almost goo/,, when the diaphragms have been fully stimulated with both theophylline and epinephrine. Various treatments with one of the agonists alone cause endogenous bindings ranging between these two extreme values. The apparent Kd value for intracellular binding according to this plot was estimated to 330 nM f 50, as compared to the apparent Kd (33-45 nM) when binding was assayed in the extracts (Fig. l and 2). Hence a difference of about one order of magnitude appears to obtain between the Kd values calculated within the cell and the 84 Intracellular Titration of Cyclic AMP-Receptor Protein Binding same constant measured with diaphragm homogenates. The double-reciprocal plot may also be used to calculate the intracellular maximal binding capacities, from its intercept with the ordinate axis. A value of 8. 9 pmol/mg protein was found which coincides with the values measured in the extracts by total cyclic [3H]AMP exchange. This discrepancy between the intracellular Kd and the Kd measured in vitro in a variety of tissue extracts including diaphragm may a t first sight seem surprising.It has however repeatedly been pointed out that cyclic AMP concentration even in the unstimulated cell was far in excess of the concentration which should result in almost maximal stimulation of protein kinases and compartmentalization of the nucleotide within the cell has usually been postulated to explain this contradiction [8,9,20]. The present work shows that despite these high intracellular concentrations of cyclic AMP, protein kinases could indeed not be fully activated, since under the same conditions, the receptor proteins appear not to be fully saturated with cyclic AMP. Concluding RemarksAs might have been expected from Equation (1) (if this reaction truly reflects intracellular conditions) a rise in cyclic AMP should be paralleled by an increase in the amount of cyclic AMP bound to receptor protein in the cell. The results reported show this indeed to be the case in the isolated rat diaphragm: when this tissue is stimulated by various agents which increase the level of cyclic AMP the amount of protein receptors endogenously saturated by cyclic AMP (R cyclic AMP) rises, as indicated in our experiments by a decrease in their ability to bind exogenously added cyclic [3H]AMP after tissue extraction.Maximal binding capacities for cyclic AMP do not seem to be affected under any circumstance. A parallel approach t o the study of this problem has been undertaken by Corbin et al. [12] and Soderling et al. [13] who investigated in adipose tissue under various stimulatory conditions, the state of activation of the catalytic subunit (C) by assaying the cyclic AMP dependence of the protein kinase in tissues extracts. These authors demonstrated that under well-defined xperimental conditions, there was a quantitative relationship between the intracellular level of cyclic AMP and the amount of the active C unit which could be separated from the complex protein kinase RC. However in their experiments high concentrations of NaCl had to be added to the extracts, since in its absence R and C tended to reassociate almost immediately, indicating that cyclic AMP is no longer bound to its receptor protein (R). The situation in various other tissue xtracts has been found to be analogous, except with skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. T his seems also to be the case for the diaphragm, since under the conditions of the present work, it has been possible to titrate for R * cyclic AMP in the crude extracts even in the absence of high salt concentrations : acccurate estimations of intracelM a r binding of cyclic AMP have been obtained and correlated with the absolute amounts of the nucleotide present in the stimulated and unstimulated cell.The binding seems t o obey simple saturation kinetics but the apparent Kd of this binding is about10 times higher as compared with the crude extracts. These results may be explained by cyclic AMP compartmentalization within the cell ; in this case, however, the simple saturation kinetics would indicate that the various pools of the cyclic nucleotide attain equilibrium very rapidly.Or else, if cyclic AMP within the cell is not compartmentalized, and if the reaction described by Equation (1) may be applied, without any modification, to intracellular equilibria, a decrease in the appare nt Kd could be merely a consequence of the dilution (about 10-fold) of the protein components during extraction of the tissue, while cyclic AMP concentrations are maintained by the addition of exogenous cyclic [3H]AMP.However these two hypotheses are certainly oversimplified, since they do not take into account factors like the intracellular concentration of the heat-stable kinase inhibitor [21,22], ATP or Mg2+ [19,23], which are known to affect cyclic AMP binding either in crude extracts or with purified protein kinase preparations. It seems impossible to decide at present which of these interpretations is most likely to reflect true intracellular conditions. It is noteworthy that the apparent Kd estimated is close to the intracehlar cyclic AMP concentration of the nstimulated tissue, a fact which should account for maximal sensitivity of the regulatory mechanisms under physiological conditions. Hormonal controls at the level of cyclic AMP-receptor protein interaction have hitherto never been described; the data reported above provide a suitable means for investigating such problems. The authors are very much indebted to Mrs Ginette Delarbre for her excellent technical assistance and to Mrs Marie-ThBrBse Crosnier for preparing the manuscript. The present work has been performed thanks to two official grants of the C. N. R. S. Paris, France: ERA No 33 and ATP No 429. 914), to a grant obtained from the D. G. R. S. T. (No 72. 7. 0135), to a generous contribution of the Fondation pour la Recherche Mf? dicale Franpise and to a participation of the CEA (Saclay, France) in the purchase of radioactive compounds. The work has been performed as a partial fulfillment of a thesis (Doctorat Bs-Sciences) submitted by L. D. -K. Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser REFERENCES 1. Robison, G. A. , Butcher, R. W. & Sutherland, E. W. (1968) Ann. Rev. Biochem. 37, 149-174. 2.Walsh, D. A. , Perkins, J. P. & Krebs, E. G. (1968) J. Biol. Chem. 243, 376 3-3765. 3. Kuo, J. F. & Greengard, P. (1969) Proc. Nut. Acad. Xci. U . S. A. 64, 1349-1355. 4. Reimann, E. M. , Brostrom, C. O. , Corbin, J. D. , King, C. A. & Krebs, E. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. 5. Tao, M, Salas, M. L. & Lipmann, F. (1970) Proc. Nut. Acad. Sci. U . S. A. 67, 408-414. 6. Gill, G. N. & Garren, L. D. (1970)Biochem. Biophys. Res. Commun. 39, 335-343. 7. Craig, J. W. , Rall, T. W. & Larner, J. 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