Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay

Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay Using rat brain membranes, buffer, atropine and 3H-QNB you will produce a displacement curve for QNB by atropine, using a filtration method to separate bound QNB from free QNB. Radioactivity on the filters will be measured by scintillation counting and, after correction for counting efficiency, will be converted into molar units from specific radioactivities. Introduction: Receptors for acetylcholine are present in many tissues and can be characterised as falling into two main types, muscarinic or nicotinic, on the basis of their ability to bind muscarine or nicotine respectively. Several substances are known that bind to the muscarinic acetylcholine receptor (mAChR): some of these are agonists (which bind and elicit a response) and some are antagonists (which bind but do not elicit a response). In general, antagonists are used to measure receptor binding as they bind with a higher affinity (lower KD) than agonists bind. In this experiment you will investigate some of the properties of mAChR in rat brain membranes by means of 3H-quinuclidinyl benzilate (3H-QNB) binding. This experiment is based upon an article by Yamamura Snyder (1974) Proc Natl Acad Sci USA 71: 1725-1729 (See course website.) Requirements: 1. Rat brain membranes store on ice. (See p for preparation method). 2. Sodium potassium phosphate (NaKP) 50 mM pH 7.4 standard 3H-QNB/NaKP assay mix (NaKP + 1.3nM 3H-QNB, 11.2 x 102 Bq/pmol high concentration 3H-QNB/NaKP assay mix (NaKP + 6.5 nM 3H-QNB, 11.2 x 102 Bq/pmol atropine solution (10 ÃŽÂ ¼M MW 290) * QNB AND ATROPINE ARE TOXIC SO HANDLE WITH CARE * 3. Small glass test tubes, micropipettes 200 ÃŽÂ ¼l (YELLOW TIPS), 1000 ÃŽÂ ¼l (BLUE TIPS), 5000 ÃŽÂ ¼l (WHITE TIPS) 4. Multiplex filtration apparatus + GF/C glass fibre filters (2.5 cm diam) + forceps 5. Scintillation mini-vials + Ultima Gold scintillant Methods: All assays have a final volume of 2.0 ml, made up of 1.5 ml 3H-QNB assay mix, 0.3 ml water or atropine. The assay is started by adding 0.2 ml membranes. The excess atropine added to the controls displaces the specific and saturable (i.e. receptor-bound) QNB leaving the non-specific, non-saturable QNB bound to the membranes. The assays are left for the appropriate length of time, stopped by adding 2.0 ml NaKP to increase the volume and filtering immediately through glass fibre filters. These are washed with NaKP and counted overnight in a scintillation counter. Day 1 1. Make up two bulk assays, one to measure total QNB binding (with water) and one to measure non-specific binding (with atropine). Set up two 50 ml conical flasks thus: A B 3H-QNB (1.3 nM) 30.0 ml 30.0 ml water 6.0 ml 0.0 ml atropine 0.0 ml 6.0 ml (this is enough for 20 assays you will do 18 assays) 2. Set up a filter tower with six GF/C filters. When you are ready, quickly add 4.0 ml swirled membranes to each flask and swirl to mix. 3. Now remove 2.0 ml aliquots to filters, three for each flask, making sure that you know which are from flask A and which from B. *USE SEPARATE PIPETTE TIPS FOR FLASKS A AND B* Note that if you contaminate the QNB solutions with atropine it will completely abolish all binding Filter quickly through fresh GF/C filters. 4. Wash each filter with 5 ml NaKP, remove filters to mini-vials, add 5 ml scintillant, invert, leave at least 1 hr, invert again and count the radioactivity in the scintillation counter. 5. Repeat steps 3 4 at times =10, 20, 30, 45 and 60 mins. 6. Using the swabs provided, take six separate samples to check for radioactive contamination, for example by rubbing gloves, bench or anything that might have been in contact with 3H-QNB. Carefully note the origin of each swab. Then put each swab into a separate vial containing 5 ml of scintillant, as before, record the treatment of each, and send them for counting. This is a standard safety procedure when dealing with radioactive chemicals. The amounts of tritium involved in this experiment are unlikely to damage your health. Nevertheless this is a useful exercise to find test your technique before you make a mistake with 32P or 125I (much more damaging). Day 2 Note that you need to take great care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your results Determine IC50 for atropine (i.e. that atropine concentration which displaces 50% of QNB binding). Take 5 small glass test tubes (1-5) and put 1200 ÃŽÂ ¼l of distilled water in each. Now add 300 ÃŽÂ ¼l of 10 ÃŽÂ ¼M atropine to Tube 1, mix well and transfer 300 ÃŽÂ ¼l to Tube 2. Mix well and transfer 300 ÃŽÂ ¼l to Tube 3. Repeat up to Tube 5. Calculate the atropine concentration in each tube. Set up 7 triplicate glass tubes (A1, A2, A3, B1 G3) as follows: Tubes 300ml of 1.3 nM QNB assay mix A 10mM atropine 1500ml B Tube 1 1500ml C Tube 2 1500ml D Tube 3 1500ml E Tube 4 1500ml F Tube 5 1500ml G distilled water 1500ml As rapidly as possible add 200ml membranes to each tube. Proceed as described in 2).4) above, using the incubation time you calculated from Day1s experiment (it should be at least 45 minutes). It is best to start the reactions in two batches, with 5 minutes between each batch to allow you time to filter the first batch before the second batch is due. Calculate the average radioactivity bound to each triplicate set of filters and convert this value into suitable units of QNB bound (nanomoles or picomoles). Plot these values against log10[atropine]. Estimate the IC50 from the midpoint of the curve and compare your result with that obtained by Yamamura Snyder. While you are waiting for the reactions to reach equilibrium, carry out a Lowry assay for protein (see p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that given in the Yamamura Snyder paper. You will be told in the class what quantities of membrane preparation to use in this assay. Day 3 Note that you need to take great care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your results Determine KD for QNB. You will make lower concentrations of QNB by diluting the standard QNB assay mix with NaKP; higher concentrations can be made from the high concentration 3H-QNB mix but this is strictly limited at 20 assays per group dont waste it. Label eight test tubes 1-8. Tube 1.3 nM QNB mix 6.5 nM QNB mix NaKP ml ml Ml 1 0 7.50 0.00 2 0 2.50 5.00 3 0 5.00 2.50 4 0 3.20 4.30 5 6.00 0.00 0.00 6 2.50 0.00 5.00 7 5.00 0.00 2.50 8 3.50 0.00 4.00 Label eight sets of triplicate tubes A1, A2, A3.H3. Add the water or atropine last. Tubes 1500 ÃŽÂ ¼l from Tube # 300 ÃŽÂ ¼l A 1 Water B 2 Water C 3 Water D 4 Water E 5 Water F 6 Water G 7 Water H 8 Water Now label a separate set of eight tubes label A4, B4à ¢Ã¢â€š ¬Ã‚ ¦H4. Set these up as the previous but add Atropine instead of water. Note that this set is not done in triplicate. Add 200 ÃŽÂ ¼l of membrane preparation to each tube. Incubate the tubes as described in 2)4) above, the incubation time being that determined on Day 1. It is best to start the reactions in two batches with 5 minutes between to allow you time to filter the first batch before the second batch is due. Calculate the average radioactivity bound to each triplicate set of filters and convert it into amounts of QNB (nano- or picomoles). Draw a straight line through the atropine controls, and subtract the values for each real or estimated atropine control from the water values and use these data to calculate the bound and free QNB values. While you are waiting for the reactions to reach equilibrium, carry out a Lowry assay for protein (p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that given in the Yamamura Snyder paper. The data from this experiment may be analysed by Scatchard analysis. This will be discussed during the following session. Further information about this and other methods of analysis can be found at: http://www.curvefit.com/introduction75.htm Dispose of your radioactive equipment and toxic chemicals in the correct places. Data analysis Questions to think about: How many dpm should be present in each assay? (Calculate this.) What is the likely nature of the non-specific binding? Comment on the rate of binding for the specific and the non-specific binding. What other methods are available for measuring receptor-ligand equilibria? If the off-rate were fast (e.g. half-life of around 1 second) what method of assaying the receptor-ligand binding might be suitable? Does the QNB concentration affect the IC50 of atropine? LOWRY ASSAY FOR PROTEIN Reagent 1: 0.5 ml copper tartrate has been mixed with 50 ml alkaline carbonate on the day of use. copper tartrate (0.1 g CuSO4.5H2O added to 0.2 g NaK tartrate in 20 ml water) alkaline carbonate (2 g NaOH in 20 ml water and adding 10 g Na2CO3, made up to 100 ml with water) Reagent 2: Commercial Folin-Ciocalteau reagent 1:1 in water Method: In a series of test tubes, add the volume of membrane announced at the start of the class and make this up to 1 ml with water. Prepare tubes containing 0, 50, 100 150 and 200 ÃŽÂ ¼g bovine serum albumin (BSA) made up to 1 ml water. The concentration of BSA you are supplied with is 1 mg.ml-1. Add 1.5 ml Reagent 1. Mix well and leave to stand for 10 min at room temperature. Add 0.3 ml Reagent 2, mix well and leave for 30 min. Read at 660 nm. Plot the data from the standard BSA tubes and calculate the protein concentration in the membranes. PREPARATION OF RAT BRAIN TISSUES Rat brain membranes for QNB binding experiment Rat brains were homogenised in 10 volumes ice-cold 0.32 M sucrose/0.1 mM PMSF with a Teflon-glass Potter homogeniser. This was centrifuged at 12000g x 10 minutes and the pellet resuspended in original volume of sucrose and frozen in aliquots. (PMSF = phenylmethylsulphonylfluoride half-life in water c. 3hr)

Feminism and Marginalization Essay

Female characters in Death of a salesman are marginalised and trivialised. Criticism from feminists reflects the lives of women and what extent they are marginalised and trivialised. Women are being relegated to a secondary level and are made unimportant as portrayed by critics, feminist and Arthur Miller in his book Death of a salesman and this can be derived from their views towards the topic. There are different reasons why and different ways in which women are made unimportant and ways in which this may be stopped if not minimized. Some feminist view marginalization and trivialization as factors brought about by equality or difference in gender or sex. They argue that women’s supposed differences from men have been used over the centuries to justify discrimination against women and their exclusion from full social and political citizenship. They argue that the constant differentiation, however has been that women have been given an inferior or secondary status in the society because of the assumed natural sexual difference pg9-10 (freedman. Feminism). For centuries difference has been the starting point of and justification for the creation of different social roles for men and women. Not only was women’s biological capacity for child birth and breast feeding and the generally lesser physical strength seen as determining their social role in the home ,occupying themselves with domestic chores and bringing up children, but it was also claimed that these biological differences made them unfit to participate in the public sphere. Women were judged to be less reasonable than men, more ruled by emotion, and thus incapable of political decision making, for example. They continue to say that the social roles and modes of behaviour that civilizations have assigned to women have kept them in an inferior position to that of men. This means that women are not like the Working classes in Marxism ideology: they have not emerged an oppressed group because of particular historical circumstances, but have always been oppressed in all forms of social organization. Ortner(1998: 21) argues: the secondary status of women in the society is one of the true universals, a pan-cultural fact. And as she goes on to explain, this secondary status of women can be explained by the fact that within multiplicity of cultural conceptions and symbolizations of women that exist and that have existed in different societies, there is a constant in the women are being closer to nature in their physiology, their social role and their psyche. Whereas women have been perceived as closer to nature, men have been perceived as closer to culture, more suited for public roles and political association. For this reason, women have been relegated to a secondary status in the society, often confined to roles in the home rather than able to accede to powerful public positions. For example Willy Loman treated his wife Linda badly, he overpowered her and he bosses her and disrespects her and s always rude to her and this is why she kept herself busy with house hold duties as any other oppressed wife would do. E. g he doesn’t give her a chance to talk when she tries to give her own opinion pg31 and he shouts at her a lot even she is doing the best she can to make situations better pg69 (Arthur Miller.  Death of a salesman) Carol Gilligan believes that the reason why women are marginalized and trivialised is because their voices have not been heard, that women have not been given a chance to air out their views because of the common culture that men are more superior. ‘Only if we can understand why their voices have been silenced, and how the dominant ideal of moral autonomy in our culture, as well as the privileged definition of the moral sphere, continue to silence women’s voices, do we have a hope of moving to a more integrated vision of ourselves of our fellow humans generalized as well as ‘concrete’ others’. Benhabib 1988:95) for example: where Linda tries to give suggestions or to air out her opinion then Willy tell her to shut up and he told her not to interrupt him. Willy does not allow her to say what she wants to say, he does not give her the opportunity to speak. This in its own way is marginalization and trivialization because it proves how men feel that everything they say is right and matters most than what women say because they feel that they are more superior than women. pg31 and pg49(Arthur Miller.  Death of a salesman) Feminist ambivalence to maternalism is based on the argument that women’s public role if framed women’s condition of oppression: the ideology of domesticity and women’s exclusion from public roles in society. The qualities and capacities make women different from men are those acquired through their condition of oppression. Pg31(Sara Goodman and Diana Mulinari. Feminist intervention in discourses on gender and development). Dorothy Smith(1987) has shown how men in position of power do not only control the world but name it and how women are excluded from the process of description and ordering the world. SHULAMITH FIRESTONE 1979:12 believes that women’s oppression is thus the primary oppression, ‘an oppression that goes back beyond history to the animal kingdom itself’, and this oppression is based on biological oppression. The effects of biology are all-pervading, and women’s inferior social position can be explained by biology- their reproductive capacity and their weakened physical condition – these biological factors being reinforced by men’s development of social structures that keep women tied to their reproductive role. pg69 (freedman.  Feminism). Examples of oppression are where Linda always tries to make situations better as way to please her husband, she takes a lot from Willy and never complains about the way he treats her. She takes all this in because she believes in pleasing her husbanding and by so doing it is shown that she is oppressed, she is not doing what she wants but what she thinks is right because she lives under the shadow of her husband and her dictatorship. E. g Linda looks out for her husband, does everything in his favour and does everything for him. E. where she asks her sons not give Willy a hard time and where she tries to make the situation batter by telling him that it was just a joke yet she know it wasn’t and she does this to protect him pg42-48(Arthur Miller. Death of a salesman) Firestone(1979) says women’s liberation is therefore ’a struggle to break free from oppressive power structure set by nature and reinforced by man’. She believes that women must be liberated th rough destruction of biological oppression and this can happen through the development of reproductive technologies that will free women from their biological reproduction capacity. In my opinion, marginalization and trivialization are phenomenons that exist in everyday life because it is believed that man are superior and women are inferior and this idea contributes thus they encourage marginalized and trivialization of women. Inequality and difference are also contributing factors because they give the impression that just because women and man are different based on their sex, women are believed to be incapable of a lot of things including decision making thus giving man the impression that they are able to rule or they are more capable of a lot than women. Oppression is also a contributing factor because it limits women from doing things to their full capacity in that they live according to what they are stipulated to do and this oppression gives man the power to belittle women and to make them unimportant. I believe that equality meaning equal opportunities to both man and women is the best solution to stopping marginalization and trivialization of women.

Local Literature

A significant amount of literature is available on inventory systems. A majority of the findings are derived from experiences in the US, Japan and Western Europe. Some inventory systems however, can be affected by variations in local conditions such as infrastructure, customs, duties, and regulations. Hence, it is essential to view such systems in an international context. To frame this research, we will examine the literature on operations in NIC countries followed by an analysis of local conditions in Thailand. A number of articles are available that examine the inventory setups in Singapore, Hong Kong, and Korea (Amsden, 1989). For example, Cheng (1988) and Hum and Ng (1995) examine the workings of Just In Time (JIT) systems in Hong Kong and Singapore, respectively. Other studies related to inventory management include facility location decisions (Sisodia, 1992; Nambiar, fielders, and Van Wassenhove, L. N, 1989; Mathews, 1997) and distributed systems. These studies indicate that Thailand is not as industrialized as the other NIC countries such as Singapore, and infrastructure shortcomings play an important role. Next, we will examine the literature on the quality of infrastructure in Thailand. Infrastructure affects both the productivity and effectiveness of manufacturing companies. It has a direct impact on the distribution of raw materials, parts, and finished goods to customers. The few studies focused on Thailand have primarily addressed infrastructural problems in Thailand (Chalamwong, Chalongphob and Wattanalee, 1994; Chalamwong, 1993). In identifying the infrastructure-related problems, Yukio (1990) calls for the Thai government to pay closer attention to transportation systems in their effort to attract more Japanese and foreign investment. Sibunruang (1986) also points to infrastructural constraints having a bearing on the development of the Thai economy. The present government, however, does realizes the importance of infrastructure for the country's overall economy. In its efforts to improve infrastructure, the Thai government has privatized telecommunication service and has started seeking help from private local and foreign companies to cope with the road shortage. The government has also approved a number of projects for the construction of highways in and around Bangkok. Furthermore, the airports in Thailand are now better managed. Although infrastructure remains a problem, there is a substantial pool of investors looking to open shop in Thailand. Information on production and inventory management practices in Thailand can help managers better understand and identify approaches that might be suitable for their companies' operations in that country. As noted earlier, no research on inventory management in Thailand has been undertaken and reported in the literature. The next section presents the methodology used in examining the types of inventory systems employed by foreign companies in Thailand.

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