3.Describe a scenario in which you would use the custom reports Essay

3.Describe a situation in which you would utilize the custom reports include under the Reports menu in the MS Project 2010 Project View - Essay Example The traditions include grants clients to create tweaked and pre-characterized reports. Moreover, in MS Project 2003, one can without much of a stretch find the report connect under the area of View | Reports. In MS Project 2007, comparative highlights are found in Report | Reports. At the point when clients pick these reports interface, an exchange box with special custom highlights springs up. The traditions highlight might be utilized when clients need to see subtleties of their arrangements in fluctuated ways (Schwalbe, 2011). A lone report may involve a unique blend of printed substance, diagrams, and tables. Not at all like numerous perspectives in MS Project, a story takes after a clear canvas where you can put any components. The highlights incorporate outlines and tables that stress on data that intrigues clients (Schwalbe, 2011). The custom highlights, comparably, encourages clients to create specific data requirements for the relating ventures. The custom element permits the execution of various capacities that would somehow be cumbersome and overwhelming. The individual highlights make report creation a straightforward

Information Communication Technology in Agriculture Essay

Data Communication Technology in Agriculture - Essay Example Notwithstanding utilizing current existing innovation creation is coming out poor, which implies there are some basic factors that are influencing it. In this paper different methodologies will be taken to sift through the key purposes of this issue. Ranchers will be met legitimately or in a roundabout way so as to get their perspectives about whether they are accepting appropriate data and information or not; or the data given to them is adequate and reasonable or not. After appropriate review Information Communication Technology (ICT) will be utilized to redress them. This paper generally focuses on various extents of ICT in the field of farming. Urbanization which straightforwardly influences the horticultural land. Before urbanization appropriate information ashore the board is important. Assume one land that can suit 100 people sufficiently however because of inappropriate land the executives just 50 people groups are getting set. It results into increment in urban zone superfluously. This issue can be settled utilizing data innovation where, individuals' propensities, tastes and ways of life are contemplated and as indicated by that ideal local location can be characterized. Climatic changes like flood dry season, climate changes likewise have impacts on foods1. The present innovation that can be utilized for advanced factual figuring so as to get thought of climatic change and dependent on that, means are taken. The most significant factor is missing ... The most significant factor is missing of appropriate information among people in general in regards to the better utilizing of assets. Here Information Communication Technology (ICT) assumes one essential job. ICT is the best apparatus for sharing information among ranchers. Setting up country system will upgrade ranchers' thought trading capacity. Aside from that utilizing remote homestead counseling framework through camera and remote, sufficient data can be conveyed. Research Questions Research proposition begins with explore question or theory. For this examination investigate questions are: 1. What are the fundamental issues that horticulture industry is confronting today 2. Will Information Communication Technology (ICT) work better for these cases 3. On the off chance that yes to address 2, at that point till what degree it will fill our need Goals The primary reason for this exploration is to perceive how data correspondence innovation will be utilized for better rural purposes. This examination will deliver the issues identified with ranchers' mindfulness and use of ICT to defeat the issues. The bearing of this work will have two three significant stages as research questions. Initial one is experiencing existing written works and devices to call attention profoundly issues, the essential factors which are answerable for that corruption and furthermore the degree they are influencing. Second stage will check whether ICT can make any improvement over the current ones. Testing and review will be done in this stage. After specialist is sure enough with ICT approach at long last, a few philosophies will be proposed to actualize the thought and afterward execution of these arrangements will be finished. Basis The above figures are models outlining the use of land

Nuns, fret not!

Nuns, fret not! Im a little nervous right now, because I have my first major exam this week (Wednesday, in chemistry), but its my favorite class (and not only because my TA is Canadian and says molar mahss) so Im less worried about it than I am about my first major paper due the day after both the exam and the season premiere of Lost. Theres no guarantee that Ill be able to concentrate that night. Instead of concentrating this weekend, I blew off steam by going on retreat with my hall (3rd East) to New Hampshire! Not all of us can go on fantasy pirate cruise ships, so we all chipped in $35 and rented cars and a cabin (apparently, MIT students can rent cars, even if theyre under 21). We danced, watched movies, and almost fell off a giant mountain. Zozer 07 smashed a fly with a spatula. What better way to get to know your hall? So a lot of you have commented about coming from mediocre schools without any particular science/math focus, and not having any research opportunities. A year ago, my comment wouldve read Whats the science olympiad? There are actually schools that focus on science and math? Can I make a baking soda volcano and submit that as research? But now? Now, my comment says Chill out, man. Also, Mr. Neha, we should throw out these bananas. Theyre a little too ripe. Its an understandable contrast, since Im writing the blog now and not applying but two of my closest friends here came from a high school that offered only 4 AP classes. My high scool was just a regular public high school, not private or magnet our biggest pride was our football team, not our academic decathalon. It just happens that way some areas are privy to more opportunities than others, and the admissions office will take that into account. I, too, had no science olympiad program readily available at my high school. I didnt have any AMC scores to submit, and I was just as terrified to submit my application with that seemingly obvious hole in the middle. So I know how easy it is to dwell on your weaknesses, and Im not saying that NOT having AIME scores will get you in, but it wont make you or break you. Its not your responsibility to cover all the weaknesses that come with your environment, because its not your fault if you cant find people who are just as excited about microbiology as you are but its also good to show initiative and look for ways around them, if you can. So look for opportunities offered outside of your school that you can take on yourself; classes offered at local colleges or online are a good place to start. And there are plenty of research opportunities out there totally unrelated to where you come from the summer after my junior year, I spent five weeks scuba diving and measuring the abundance of Diadema antillarum, the spiny sea urchin, inside and outside of the marine protected areas of South Caicos through the School for Field Studies. I was one of three high school students there, and the knowledge and maturity I gained from that experience is something Id never have found at my high school it put me in a place where I was utterly uncomfortable and forced me to adapt to my surroundings. But theres no set formula to one application MIT is looking for more than that. The important thing to remember when youre applying, whether you have too much to write about or nothing at all, is that youre being evaluated on you, as a person, and whether you as a person fits at MIT, not whether or not you come from a magnet school or are homeschooled. Because after this whole thing is over, youll no longer be a part of that mediocre school, but youll very much still be you as a person. The only difference is youll be a freshman in college wondering why your bananas, too, are so very ripe. It happens to the best of us. Post Tagged #Next House

Cost of Poor Quality in Banking - 3462 Words

Industrial research report COST OF POOR QUALITY SUBMITED TO: MR. AZHAR NISAR LETTER OF TRANSMITTAL Mr. At Gulistan-e-Johur Karachi. RESEARCH VISIT BY STUDENTS Dear Sir, Assalam o alikum Bahria University is federally accredited University based at Islamabad with its campus at Karachi. University is educating is students in the fields the of management science, computer, Engineering, Medical and Dental Surgery. Management science students are guided to carry out subject based research in various business and corporate organizations. This enhances their abilities to think independently and resolve the management problems logically. Some students of BBA-7 have selected your bank to carry out research with reference†¦show more content†¦Particular attention should be given to the cost of poor quality and to customers views about the relative importance of the attributes of service. If the cost of quality is high, looking through the Six Sigma, the cost of poor quality is still higher. Companies bear a huge cost of about 9-16 percent of their revenues on problem solving. COPQ can take in the following forms: †¢ cost generated from producing defective material †¢ cost involved in fulfilling the gap between the desired and actual product or service quality †¢ cost of lost opportunity due to the loss of resources used in fixing the defect, including all the labor cost, rework cost, disposition costs and material costs that have been added to the unit up to the point of rejection †¢ appraisal cost if there is an inspection point Banks are fined for failing to provide accurate transaction reports to the FSA and for serious weaknesses in systems and controls in relation to transaction reporting. One there is the reputational damage that comes from being fined and then there is the cost of correcting errors and fixing the defective processes. It is seen that companies ignore the cost quality analysis, overlooking its potential significance and end up paying failure cost. 1†¦ how does employee involvement support in TQM. If u r required develop program. What elements would u include? 2†¦ think of a team u have been on recently. It could be aShow MoreRelatedBenefits Of Credit From Multiple Lenders1148 Words   |  5 Pagesconsidering the cost incurred when it is denied credit by its bank for reasons. As an illustration, a temporary liquidity shortage leads the bank to be forced to deny credit even to its loyal borrowers. Hence, given these possibility and risk of not being able to raise funding from an alternative bank for the first time, it may be worth to initiate and maintain multiple lending relationships despite the costs is needed. 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Thus I believe the study of these banks will provide the fair condition of banking sector of the country. The banks of

Essay Abortion - 1041 Words

Abortion is never an easy decision, in fact its one of America’s most controversial issues in today’s reality, but women have none the less been making that choice for thousands of years. Studies show that about 43% of American women will have one or more abortions during their lifetime, and womens centers and hospitals perform more than a million abortions on an annual basis. Women have many reasons for not wanting to be pregnant including age, marital status, economic status, and the circumstances of their pregnancy, and thus seek out an abortion. Although many citizens view abortion as an immoral act of brutality and strongly contest its usage, others believe the choice belongs solely to the mother and the mother alone. The main†¦show more content†¦Thousands died. Tens of thousands were mutilated. All were forced to behave as if they were criminals. (Durrett 126) Another key issue in regards to abortion is whether or not a raped woman should continue and bare the child or chose to abort the fetus. Pro-lifers believe that the unborn child has the right to live and even if the woman faces an unwanted pregnancy, she should carry the child to term and make the best of her situation with the help available to her and her family. They further believe an abortion only traumatizes the raped woman further, when she realizes she has killed her own child. Through an abortion, the mother becomes the aggressor and ultimately murders a future child. (Brennan 26) Under pro choice views, during rape the woman does not give consent to participate in sexual activities and therefore can not be held liable for the pregnancy forced onto her by the criminal acts of injustice done to her. In this circumstance, abortion would not be considered murder and by doing so the victim would be able to retain a normal life once again. Now, if women are forced to carry unwant ed pregnancies to term, the result would be an unwanted child. (Sloan 27-34) These children epitomize societys most awful cases, often neglected, unloved, brutalized, and more often than not, simply abandoned along deserted ally ways, or in dumpsters or even on the sides of freeways. When they grow up, these children are oftenShow MoreRelatedAbortion : Abortion And Abortion998 Words   |  4 PagesAbortion Abortion is defined in several ways all of which stop a pregnancy. There are different ways of abortion, which are spontaneous abortion, surgical abortion, and medical abortion. Abortion has been arguable topic for decades. One can neither believe abortion to be good nor bad. The idea of individuality and human life is not quite the same. 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Titration Journal Free Essays

E r J. Biochem. 40,177-185 (1973) u. We will write a custom essay sample on Titration Journal or any similar topic only for you Order Now 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 subunit (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 intracellular 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-epinephrine 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 extract. 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, acetate 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 lev el 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 different 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 Fraction 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 acid 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 activity 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 extracts 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 (meth od 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 diaphragms 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 were 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 fractionati on 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 measure 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 ext racts (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. Biochem. 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, 182 Intracellular 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/, trichl oroacetic 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 with 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 binding 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 quant itatively 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 A M 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 epinephrine 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 bindin g 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 in tracellular 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 pm ol/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 Remarks As 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 wit h skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. This 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 apparent 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, 3763-3765. 3. Kuo, J. F. Greengard, P. (1969) Proc. Nut. Acad. Xci. U . S. A. 64, 1349-1355. 4. Reimann, E. M. , Brostrom, C. O. , Corbin, J. D. , King, C. A. Krebs, E. G. (1971) Biochem. Biophys. Res. Commun. 42, 187-194. 5. Tao, M, Salas, M. L. Lipmann, F. (1970) Proc. Nut. Acad. Sci. U . S. A. 67, 408-414. 6. Gill, G. N. Garren, L. D. (1970)Biochem. Biophys. Res. Commun. 39, 335-343. 7. Craig, J. W. , Rall, T. W. Larner, J. (1969) Biochim. Biophys. Acta, 177, 213-219. 8. Stuil, J. Mayer, S. E. (1971) J. Biol. Chem. 246, 5716-5723. 9. Schaeffer, L. D. , Chenoweth, M. Dunn, A. (1969) Biochim. Biophys. Acta, 192, 292-303. 10. Miller, T. B. , Exton, J. H. Park, C. R. (1971) J. Biol. Chem. 246, 3672-3678. 11. Harbon, S. Clauser, H. (1971) Biochem. Biophys. Res. Commun. 44, 1496-1503. 12. Corbin, J. D. , Soderling, T. R. Park, C. R. (1973) J. Biol. Chem. 248. 1813-1821. 185 13. Soderling, T. R. , Corbin, J. D. Park, C. R. (1973) J. Biol. Chem. 248, 1822-1829. 14. Gill, G. N. Garren, L. D. (1969) Proc. Nut. A d . Sci. U. 8. A. 63, 512-519. 5. Walton, G. M. Garren, L. D. (1970) Biochemistry, 9, 4223-4229. 16. Gilman, A. G. (1970) Proc. Nut. Acad. Sci. U. 8. A. 67, 305-3 12. 17. Do Khac, L. , Harbon, S. Clauser, H. (1973) Ninth Int. Congr. Biochem. p. 354. 18. Lowry, 0. H. , Rosebrough, N. J. , Farr, A. L. Randall, R. J. (1954) J. Biol. Chem. 193, 265-275. 19. Wilchek, M. , Salomon, Y. , Lowe, M. Selinzer, Z. (1971)Biochem. Biophys. Res. Commun. 45,1177-1184. 20. Chambaut. A. M. , Lerav, F. Hanoune, J. (1971)PEBS . . â€Å". Lett. 15,’328-334. Walsh, D. A. , Ashby, C. D. , Gonzalez, C. , Calkines, D. 21. Fisher. E. H. Krebs. E. G. (1971)J. Biol. Chem. 246, i977-1985. 22. Ashby, C. D. Walsh, D. A. (1973) J. Biol. Chem. 248, 1255-1261. 23. Haddox. M. K. , Newton, N. E. , Hartler, D. K. Goldberg, N. D. (1972) Biochem. Biophys. Res. Commun. 47,-653-661. 24. Klotz, I. M. (195 3)in The Proteins (Neurath, H. Bailey, K. , eds) p. 772, Academic Press, New York. 25. Cleland, W. W. (1967) Advan. Enzymol. 29, 1. , L. Do Khac, S. Harbon and H. J. Clauser, Institut de Biochimie, Universit6 de Paris-Sud, BLtiment 432, F-91405 Orsay, France Eur. J. Biochem. 40 (1973) How to cite Titration Journal, Papers

Elements of Compensation Packages

Question: Write an essay on Elements of Compensation Packages. Answer: From the given case, it is notable that Bill Strong, who has been the Chief Executive Officer (CEO) and founding Director of the Strong Built Construction Company, has designated Susan Bold as the companys new Chief Financial Officer (CFO). Currently, the industry has witnessed a state of depression however this has not majorly affected the company. Despite this, a conducted survey disclosed that the company is facing an issue regarding poor employee motivation. The present approach of rewarding employees involves providing them with company shares. Bill believes that the approach of conventional agency theory motivates employees by providing them with monetary benefits. However, Susan has a different perception, who believes that intrinsic considerations are necessary while rewarding employees. Contextually, this part of the assignment will create a report from the perspective of Susans assistant. Elements of Compensation Packages There are different elements of compensation packages that the company can consider while rewarding its employees. In this regard, Gerhart, Minkof and Olsen (1995) suggested the base salary as one of the decisive aspects of employee compensation packages, which is the fixed salary paid based on the work performed. Moreover, according to Fogleman and McCorkle (2009), the compensation package to the employee includes bonuses, which may be on annual, half-yearly or quarterly basis. This largely motivates employees to perform well and contribute to the attainment of organizational goal. Besides, bonuses are provided for outstanding performances. According to the report published by Labour Department (2015), for employees motivation, companies provide compensations for health and wellness, which include medical, vision, dental and other health related assistance. Gerhart, Minkof and Olsen (1995) also suggested that several companies provide life and accident insurances to its employees, w herein monetary and other assistances are provided for accidental death of the respective employees, long-term disability and dismemberment. These types of compensations largely motivate employees and influence them to be loyal to the company. Furthermore, for attracting employees, several companies provide free travelling opportunities (Labour Department, 2015). In addition, the compensations such as food subsidy, internet services, cell phones, membership in premium clubs, and education funding among others provided to the employees motivate them to perform well (Gerhart, Minkof and Olsen, 1995). Thus, it is apparent that there are several different elements of compensation, which the Strong Built Construction Company can provide to motivate its employees. Assumptions of Traditional Agency Theory and Their Influence on Compensation There are certain key assumptions of traditional agency theory, which largely influence the concept of compensation. In this regard, Kivisto (2007) mentioned human assumptions as one of the forms of the theory, wherein most of the people within an organization are conscious about self-interest. It is assumed that the manager, the owner, as well as the employees of Strong Built Construction Company have self-interest, which in turn largely influences compensation. Human assumption also indicates the risk aversion characteristics of the manager as well as the employees, which considerably affect their performance, thereby resulting into low compensation. Human assumption also involves the bounded rationality, which suggests that the manager and the owner make decisions based on the limited information that they possess regarding the employees. This in turn largely affects their compensation (Rodriguez, Gomez-Meizia and Wiseman, 2012; Kivisto, 2007). The other assumption of the traditional agency theory as mentioned by Kivisto (2007) is the organizational assumption, which indicates the persistence of goal conflicts among the internal stakeholders of the company. In the current company, there is a possibility that the owner, manager, and the employees have different individual goals. The employees might perform differently with respect to organisational goal, which in turn affects their compensation. Moreover, organisational assumption also includes the information irregularity between the owner and the manager. This affects the decision making process, thereby hampering the employees compensation benefits to a large extent (Rodriguez, Gomez-Meizia and Wiseman, 2012; Kivisto, 2007). Difference and Relationship between Extrinsic and Intrinsic Motivation Ims, Pedersen and Zsolnai (2014) suggested of the relationship and difference between intrinsic and extrinsic motivation. In this context, the relationship between both these forms of motivation is to provide certain rewards to the individuals, which in turn encourages them to perform well. In the current organization, both these types of motivation encourage the employees to participate in the organizational decision-making and share common goal of interest. Thus, the relation between these motivations is to ensure mutual benefit of the employees as well as the organisation. However, extrinsic motivation occurs to gratify the external needs of an individual. On the other hand, intrinsic motivation attempts to satisfy the personal needs and desires of an individual. Extrinsic motivation factors are largely similar for different individuals. However, intrinsic motivation factor differs from individual to individual. Therefore, identification of intrinsic motivation factor is difficult in comparison with the extrinsic factors of motivation (Ims, Pedersen and Zsolnai, 2014; Sansone Harackiewicz, 2000). Employees Attitude Influencing Compensation Package Pepper and Gore (2014) mentioned that there are two types of employee attitude within an organization, which are risk adverse and risk seeking. In relation to the concerned organization, the employees with an attitude of risk adverseness avoid taking additional responsibilities. This in turn affects their compensation. Besides, the risk adverse attitude of employees also discourages the manager as well as the owner to keep trust on them, which eventually affects their professional growth. On the other hand, the manager and the owner of the organization are highly encouraged by observing the risk-seeking attitude of the employees. Correspondingly, they trust the respective employees and offer higher responsibilities. Subsequently, they receive higher professional growth, which significantly increases their desired compensation (Pepper and Gore, 2014; Kumar, 2010). Time-Period Influencing Employees Desire for Benefit Pepper and Gore (2014) suggested that the time when employees attain compensation benefit is a very decisive factor. Appropriate time of receiving the compensation by the employees largely influences their desire of availing the benefit. Concentrating on the given scenario, if the employees receive health or medical insurance support at the time of need, they will be highly satisfied. Besides, bonuses at the time of festive-occasions influence the employees to have greater desire for the benefit. Employees also desire for the base salary benefit to be received at the specified date of every month. On-time availability of the benefits largely motivates the employees, which in turn influences them to be loyal to the company and attain their desired benefits (Pepper and Gore, 2014). Role of Fair Consideration on Compensation Pepper and Gore (2014) mentioned that fair consideration to the employees of an organization plays one of the major roles in determining appropriate compensation. It is applicable to the given construction company as well. In this regard, fair consideration plays a significant role in ensuring desirable compensation, which in turn motivates employees to perform better. This encourages them to attain their individual objectives aligned with the organisational goal. The employees, who are not performing expectedly, are motivated because of fair consideration. Thus, fair consideration has the role of improving the employee productivity, which in turn ensures their professional growth, thereby determining greater amount of compensation (Greene, 2010; OECD, 2009). Executive Compensation Committee Benefiting in Determining Compensation The introduction of Executive Compensation Committee in Strong Built Construction Company will be of significant benefit in determining the appropriate compensation. The reason behind this as mentioned by Hermanson, Tompkins, Veliyath and Ye (2012) is that it ensures long-term incentive plans to the employees, which eventually contributes to employee loyalty in the organisation. Besides, it considers appropriate compensation philosophy, which involves the attainment of organisational goals, retention of employees, connection of the employee compensation with organisational goals, and providing fair as well as reasonable compensation. The committee also considers suitable documentation process, which ensures the determination of appropriate compensation (Sirkin Cagney, 2015; Reda, Reifler Stevens, 2014). Structuring of Executive Compensation Committee Strong Built Construction Company to obtain the best outcomes with respect to compensation requires introducing an Executive Compensation Committee. In this regard, for appropriate structuring of the committee, the organisation requires considering some of the decisive aspects. One such aspect as stated by Hermanson, Tompkins, Veliyath and Ye (2012) is the size of the committee, which typically comprises three to five individuals. Moreover, for appropriate structuring of the committee, the board of directors requires appropriate selection of the committee members. The selection is based on the qualification and knowledge of the members, their experience in the professional field along with adequate training (Sirkin Cagney, 2015; Reda, Reifler Stevens, 2014). Conclusion From the above discussion, it is apparent that for Strong Built Construction Company, determining appropriate compensation is necessary. Thus, it is recommended that the organisation apart from providing shares to the employees must offer compensations such as bonuses, health and medical insurance and other facilities. Moreover, it is also recommended that the company must focus on providing intrinsic motivation to the risk-seeking employees. It should also consider appropriate time and fair consideration while providing compensations. Furthermore, it should introduce an Executive Compensation Committee for determining appropriate compensation. Aim of the Research The mandatory implementation of revised ISA 700 Auditors report attempts to lessen the Audit Expectation Gap. Thus, considering this aspect, the research by Gold, Gronewold and Pott (2012) attempted to examine its effectiveness. Elaborately, the research aimed towards evaluating the present state of the audit expectation gap in Germany with respect to that of the revised ISA 700 auditors report through several experiments. The research also aimed at taking the support from several business students as unsophisticated users and business analysts as sophisticated clients, along with the auditors who have considerable experience performing in the field of work. These individual groups were asked to evaluate an unqualified ISA 700 auditors report. They were also correspondingly requested to provide response regarding the roles as well as responsibilities of the management and of the auditors along with the reliability of audit reports. Purpose of Manipulation Check Gold, Gronewold and Pott (2012) perceived that the respondents might change their response after reading the subsequent questions. Thus, the purpose behind conducting the manipulation check is to ensure that the respondents do not go back to the previous question of the dependent variables after reading the corresponding questions. Thus, manipulation check ensured true and appropriate response of the provided questions. Research Aim The aim of the study by Agyei, Aye and Owusu-Yeboah (2013) is to evaluate the persistence of Audit Expectation Gap in Ghana. This assessment is made from the perspective of stockbrokers and auditors. The rationale behind conducting this particular research is that there have been no previously conducted studies relating to the Audit Expectation Gap in Ghana. On the other hand, the conducted study by Okafor and Otalor (2013) aimed at determining whether people are familiar with the regulatory, legislative and professional statements of the auditors. It also aimed to determine whether the responsibilities and duties of the auditors are appropriate to conduct. Research Approaches in the Studies Agyei, Aye and Owusu-Yeboah (2013) in their study to determine the audit expectation gap in Ghana incorporated qualitative approach, wherein calculation of no quantitative data has been conducted. The qualitative approach of measuring the audit expectation gap in Ghana has been done through the logical interpretation of collected data along with the concept generated from the literature sources. On the other hand, the research conducted by Okafor and Otalor (2013) integrated mixed approach, which involved both quantitative as well as qualitative analysis. The integration of mixed approach supported in cross-validation of data. Thus, noting both the approaches used in the respective studies, it can be evaluated that the mixed approach used in Okafor and Otalor (2013) is more rigorous. Owing to such approach, the results derived are believed to be more reliable, valid and relevant. Research Participants in the Studies The study by Agyei, Aye and Owusu-Yeboah (2013) selected 20 stockbrokers along with 20 auditors as the participants for questionnaire survey. These participants were selected based on purposive sampling, believing that the stockbrokers and the auditors have provided valid, reliable, and relevant information. On the other hand, the research conducted by Okafor and Otalor (2013) involved randomly selected teachers and students from the University of Benin, Ambrose Alli University, Benson Idahosa University, Ekpoma, the investing public along with the Accountants in Practice in Edo State. On a comparing note, it can be evaluated that although the study by Okafor and Otalor (2013) integrated random sampling to reduce biasness, the research by Agyei, Aye and Owusu-Yeboah (2013) is more rigour with respect to selection of research participation. The reason behind such is that the involved participants, i.e. auditors and stockbrokers have adequate and appropriate knowledge regarding audit e xpectation gap. Response Rate in the Studies The respondents involved in the research conducted by Agyei, Aye and Owusu-Yeboah (2013) was a total of 40, which comprised 20 stockbrokers and 20 auditors. Although the response rate was not significantly high in the study, the participants involved are extensively proficient in providing appropriate responses. However, the questionnaire survey conducted by Okafor and Otalor (2013) involved 130 respondents. In spite of the returned 94 questionnaire from the 130 respondents, which accounted to 72%, the value is sufficient to give unbiased result as a whole. Data Analysis in the Studies In the study by Agyei, Aye and Owusu-Yeboah (2013), the collected primary and secondary data are analysed through qualitative method. Elaborately, logical interpretation technique was used to analyse the collected data. The primary data that were collected from questionnaire survey were analysed and justified with the support of the concept developed from literature sources. On the other hand, in the research by Okafor and Otalor (2013), mixed analysis has been incorporated, which involve both quantitative and qualitative methods. The collected primary data were analysed through ANOVA, logit and probit, along with multiple regression, which supported in hypotheses testing. The collected primary data were also analysed from the concept generated from secondary sources. The use of both quantitative and qualitative method for analysis supported in cross validation of data, which thereby ensured greater reliability, validity and relevancy of research outcome. Thus, considering these aspe cts, it can be evaluated that the data analysis method used in Okafor and Otalor (2013) is more rigor. Moreover, the use of comparatively greater number of secondary sources in the study also ensured its analysis to be more rigorous. Two Other Significant Flaws in the Studies The study conducted by Agyei, Aye and Owusu-Yeboah (2013) involved only stockbrokers and auditors as participants. In this regard, it can be commented that there are several users of financial statements, which were not involved in the study. Besides, the study did not incorporate quantitative analysis, which could have led to a further comprehensive research. Okafor and Otalor (2013), in their research have not included open-ended questions and interview. The other flaw in the study is that there were several secondary data considered that were published decades back. This limits its relevancy to the current day context. References Agyei, A, Aye, BK Owusu-Yeboah, E 2013, 'An assessment of audit expectation gap in Ghana', Int. J. Acad. Res. Account., Financ. Manage. Sci, vol. 3, no. 4, pp. 112-8. Fogleman, SL, McCorkle, D 2009. Human Resource Management: Employee compensation guide, AgriLife Extension, pp. 1-4. 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