Binding of QNB and Atropine to Muscarinic Acetylcholine

Binding of QNB and Atropine to Muscarinic Acetylcholine Cholinergic relates to the responses in various systems to the neuro-transmitter molecule Acetycholine (ACh). They are the protein that are permanently attached to the biological membrane or the integral membrane protein (IMP). If the set of response is seen where Ach is a normal transmitter it is seen that they are grouped based on nicotinic acetylcholine receptors (nAChR) that respond to nicotine, and muscarinic acetylcholine receptors (mAChR) that bind muscarine. These Nicotine and muscarine are extrinsic molecules that get the same response but with different sensitivity. Drugs that bind to muscarinic receptors are classified based on Agonists (which activate the neuronal receptor and produce a response) Antagonists (which do not activate the receptor and block the agonist binding site) Antagonists are now used to study the drug-receptor binding as they bind with a higher affinity (i.e lower dissociation constant kd) when compared with agonists Pharmacology studies have shown that antagonists have higher affinity but no efficacy to their cognate receptors. They intervene their effect by going and binding to the active site or to allosteric sites on the receptor. They can also go and bind to unique binding sites that do not participate in biological regulation of any receptor activity. The activity that antagonist causes may be reversible or irreversible, depending on the long life of the antagonist-receptor complex. Studies have shown that 3-Quinuclinidyl benzilate (QNB) is a potent muscarinic antagonist in CNS (central nervous system) and peripheral tissues. QNB shows specific binding to the receptor of interest it binds. It can also bind to other sites of the membrane and these can cause changes. We can measure specific binding by filtering radioactive 3H-QNB and then measuring the amount of QNB. To measure non-specific binding, Atropine is used to displace QNB from the specific sites, while the non-specifically bound QNB remains and can be quantified by measuring radioactivity. (Source: Yamamura et al. May 1974) Overview of the experiment QNB is carried out in a radioactive binding assay where the concentration of QNB that is specific bound without atropine and QNB that is non-specifically bound with atropine is measured over successive interval of time. It is allowed to incubate so as for binding site to reach saturation is allowed where the equilibrium is reached. After this any further increase incubation time does not cause the amount of QNB bound to change. This QNB bound to the membrane is measured. By calculating the incubation time, IC50 of atropine is measured by measuring the atropine at which 50% of bound QNB is displaced. Amount of free QNB when 50% of bound QNB is displaced is used to measure the dissociation constant (Kd). Materials and Methods Determination of QNB specific and non-specific binding Two bulk assays was carried out To measure QNB binding (in the presence of water) To measure non specific binding (with the presence of atropine) There were two conical flask taken A and B. Tube A was added with 30 ml of 1.3 nM 3H-QNB and 6ml water. And to the flask B flask B, 30 ml 3H-QNB and 6ml atropine was added. S filter tower is then set with 6 GF/C filters and 4.0 ml of rat membrane was added to each flask and the flask were swirled to mix well. 2ml aliquots from A flask (A1, A2, A3) and (B1, B2, B3) from the B flask were produced and were run through fresh GF/C filters. Each of the filters was then washed to remove mini-vials, and then 5 ml scintillant was added and was left for at least an hour. After a hour the radioactivity was counted in the scintilliant counter. This protocol was repeated for a couple of more time to produce triplicates at the time interval of 10, 20, 30, 45 and 60 min. Determination of IC50 for atropine Five glass test tubes having 1200 ÃŽÂ ¼l of distilled water in each was taken. To the test tube 1, 300 ÃŽÂ ¼l of 10 10 ÃŽÂ ¼M atropine was added and was mixed well. 300 ÃŽÂ ¼l of the solution was added to tube 2 and mixed well. The same method is carried out for a series of dilutions to be done in tube 3 to 5. Atropine concentration in each tube is calculated. Seven triplicate tubes (A1, A2, A3à ¢Ã¢â€š ¬Ã‚ ¦G1, G2, G3) are made each containing 1500 ÃŽÂ ¼l of 1.3nM QNB assay and the tubes are mixed well. 300 ÃŽÂ ¼l of 10 ÃŽÂ ¼M atropine was added to the three tubes of A and three B tubes were added with 300 ÃŽÂ ¼l of solution from tube 1. The dilution process was carried out for tubes C, D, E, F from tube 2, tube 3, tube 4 and tube 5 respectively. To tubes G, 300 ÃŽÂ ¼l of distilled water was added instead. 200 ÃŽÂ ¼l membrane was then added quickly to all the tubes. The 21 tubes were then left for incubation for 45 min and the radioactivity was then measured. Determination of concentration of protein using Lowry Assay Test tubes were prepared that contained 0, 50, 100, 150 and 200 ÃŽÂ ¼g BSA (Bovine serum albumin) made up to 1 ml with water. A 6th tube was taken that had 50 ÃŽÂ ¼l of membrane that was made up to 1ml with water. 1.5ml of reagent 1 that contains 0.5 ml copper tartrate + 50ml alkaline carbonate was added and mixed well and let to stand for 10 min at room temperature. Then 0.3 ml of reagent 2 that contains Commercial Folin-Ciocalteau reagent was added to the tubes and mixed well. The tubes were then left for incubation for 30 min. Absorbance or optical density was read at 660nm. Determination of kd for QNB Eight test tube was taken, four containing low QNB concentration (1.3nM QNB mix) and four tubes containing high QNB concentration (6.5nM QNB mix). Tubes 1 to 4 were added with 7.50 ml, 2.50 ml, 5 ml and 3.2 ml of 6.5 nM QNB mix respectively. Lower concentration of QNB is made by diluting the standard QNB assay mix with NaKP solution. These tubes are labelled 1-8. The solution of tube 1-8, of about 1500 ÃŽÂ ¼l each was added to the triplicate tubes (A1, A2, A3, H1, H2, H3) respectively. Solution of tube 1 is added to tubes A, Tube 2 to B tubes till tube 8 to tubes H. 300 ÃŽÂ ¼l water + 200 ÃŽÂ ¼l membrane was then added to all tubes. For tubes A4-H4, 300 ÃŽÂ ¼l Atropine plus (Tube 1-8) respectively plus 200 ÃŽÂ ¼l membranes was added. Radioactivity was measured in all tube. A lowry assay was also carried out. RESULT AND DISCUSSION Here in the graph the values are plotted for QNB bound with atropine (with as show in the graph), QNB bound without atropine (Without as shown in the graph) and Corrected vales are obtained by subtracting QNB bound with atropine from the QNB bound without atropine (corrected as shown in the graph ) against time. Here QNB bound without atropine is total amount of QNB bound to the receptor; QNB bound with atropine is the Non-specific binding of QNB to the receptor and corrected is the specific binding of QNB to the receptor. After a particular time of incubation receptors reach equilibrium, where no more binding of QNB takes place to the binding sites. At this point when no more binding of QNB takes place the plateau is formed in the graph showing saturation. This incubation time is approximately 45 min as shown by the graph reaching the plateau. The graph shows us that with and corrected points of the graph forms a plateau after reaching incubation time of approximately 45 min. If an addition incubation time was taken after 60 min we would have got a plateau for without graph also showing us a plateau. The graph shows that the cmp value increase over time after which when reaching a particular time no more binding occurs thus forming a plateau showing the saturation or equilibrium has reached. Small decline in the graph can be seen at time 30 to 45 min, this could have been due to experimental errors. The errors could have been caused during pipetting, in proper vacuum, formation of bubbles, adding samples properly between time intervals etc. This can be avoided by more careful handling of the instrument and doing a initial check up for errors so as to not cause changes in the experiments result. Taking the above data into consideration we have chosen 45 min as incubation time for determining IC50 of atropine. This is because, saturation of binding sites is achieved and no further unbinding of QNB also occurs, as the off-rate or reaction constant of QNB unbinding is very low. So there is no further change in the amount of bound QNB and hence this incubation time is considered appropriate. By serial dilution different concentration of atropine was prepared. The graph shows us that the amount to QNB bound to the receptor of the membrane reduces with increase in concentration. This happens because atropine is a competitive binder and binds competitively with specific sites to the receptor. The amount of QNB specifically bound will be inversely proportional with atropine concentration. Half maximal inhibitory concentration (IC50)  is a measure of how effective a compound is in inhibiting biological or biochemical function. This is a quantitative measure that let us know how much concentration of the drug or biological substance (inhibitor) is required to inhibit a given biological process by half. So we are calculating the IC50 of atropine to determine its potency. It is calculated by taking atropine concentration at which 50% QNB is displaced. The IC 50 value was found to be 0.0008912 ÃŽÂ ¼M. This shows that atropine is a drug with good potency. Ic 50 does not directly discuss the binding constant so we cannot compare the binding affinity of QNB and receptor. Lowarys assay Lowrys assay was carried out for determining the concentration of membrane protein. First different concentration of BSA was used and we generated a graph for it, taking concentration and OD. The membrane protein was then checked for absorbance and was found to be 0.322. Using the linear regression equation and the absorbance, concentration of the membrane protein was found to be 0.803 mg/ml. This test was done for another membrane protein sample. The absorbance of the membrane was 0.27. Again using the regression equation and the absorbance, concentration of the membrane protein was found to be 0.293529412 mg/ml. Determination of Kd: Kd is -1/m and was the equation was used is y = -8499.6x 1.3669. the kd is used to define the affinity between the drug and the protein . the value of Bmax was 0.001161 nm.

Culture of Pakistan Essay

The 17th century Badshahi Mosque built by Mughal emperor Aurangzeb in Lahore The society and culture of Pakistan (Urdu: Ø «Ã™â€šÃ˜ §Ã™ Ã˜ ª Ù ¾Ã˜ §Ãš ©Ã˜ ³Ã˜ ªÃ˜ §Ã™â€ Ã¢â‚¬Å½) comprises numerous diverse cultures and ethnic groups: the Punjabis, Kashmiris, Sindhis in east, Muhajirs, Makrani in the south; Baloch and Pashtun in the west; and the ancient Dardic, Wakhi, and Burusho communities in the north. These Pakistani cultures have been greatly influenced by many of the surrounding countries’ cultures, such as the Turkic peoples, Persian, Arab, and other South Asian ethnic groups of the Subcontinent, Central Asia and the Middle East. In ancient times, Pakistan was a major cultural hub. Many cultural practices and great monuments have been inherited from the time of the ancient rulers of the region. One of the greatest cultural influences was that of the Persian Empire, of which Pakistan was a part. In fact, the Pakistani satraps were at one time the richest and most produc tive of the massive Persian Empire. Other key influences include the Afghan Empire, Mughal Empire and later, the short-lived but influential, the British Empire. Pakistan has a cultural and ethnic background going back to the Indus Valley Civilization, which existed from 2800–1800 B.C., and was remarkable for its ordered cities, advanced sanitation, excellent roads, and uniquely structured society. Pakistan has been invaded many times in the past, and has been occupied and settled by many different peoples, each of whom have left their imprint on the current inhabitants of the country. Some of the largest groups were the Proto-Indo-Aryans, of which Sindhis and Punjabis descend from and later Iranic peoples which the Baloch and Pashtuns descend from. Other less significant ones include the Greeks, Scythians, Persians, White Huns, Arabs, Turks, Mongols, Buddhists, and other Eurasian groups, up to and including the British, who left in the late 1940s. The region has formed a distinct cultural unit within the main cultural complex of South Asia, the Middle East and Central Asia from the earliest times, and is analogous to Turkey’s position in Eurasia.[1] There are differences in culture among the different ethnic groups in matters such as dress, food, and religion, especially where pre-Islamic customs differ from Islamic practices. Their cultural origins also reveal influences from far afield, including Tibet, Nepal, India, and eastern Afghanistan. All groups show varying degrees of influence from Persia, Turkestan and Hellenistic Greece. Pakistan was the first region of South Asia to receive the full impact of Islam and has developed a distinct Islamic identity, historically different from areas further west.[1] Ancient sites in Pakistan include: Zoroastrian Fire temples, Islamic centres, shi’a shrines/Sufi shrines, Buddhist temples, Sikh, Hindu, and pagan temples and shrines, gardens, tombs, palaces, monuments, and Mughal and Indo-Saracenic buildings. Sculpture is dominated by Greco-Buddhist friezes, and crafts by ceramics, jewellery, silk goods and engraved woodwork and metalwork. Pakistani society is largely multilingual, multi-ethnic and multicultural. Though cultures within the country differ to some extent, more similarities than differences can be found, as most Pakistanis are mainly of Aryan heritage or have coexisted side by side along the Indus River for several thousand years, or both. However, over 60 years of integration, a distinctive â€Å"Pakistani† culture has sprung up, especially in the urban areas where many of the diverse ethnic groups have coexisted and ithe country now having a literacy rate of 55%, up from 3% at the time of independence. Traditional family values are highly respected and considered sacred, although urban families increasingly form nuclear families, owing to socio-economic constraints imposed by the traditional culture of the extended family. The past few decades have seen emergence of a middle class in cities such as Karachi, Lahore, Rawalpindi, Hyderabad, Quetta, Faisalabad, Sukkur, Peshawar, Sialkot, Abbottabad, and Multan. Rural areas of Pakistan are regarded as more conservative, and are dominated by regional tribal customs dating back hundreds if not thousands of years. â€Å"Pakistan’s culture is again unique like the rest of the country. Pakistan’s geography is the meeting point of South Asia, Central Asia and West Asia/Gulf. Its culture could be termed as a combination of sub continental, Islamic, Regional, English, and more recently global influences. Let us consider them piecemeal. The newly born Pakistan had to have a sub continental leaning, having been a part of for last 5000 years of its civilization. However, the Indus Valley, present day Pakistan, culture was different from the rest of North India or South India†. (Quoted Pakistan’s Identity, History and Culture, from the famous book Gwadar on the Global Chessboard by Nadir Mir)

Modernizing Mental Healthcare And The Juvenile Justice...

Modernizing Mental Healthcare in The Juvenile Justice System Rhoshunda Ellis Walden University Modernizing Mental Healthcare in The Juvenile Justice System Introduction As a Human Services Professional with a background in criminal justice, this article will focus on accessing and helping juvenile offenders in the United States struggling with mental health disorders. For sentenced juveniles with behavioral problems and concerns of mental health, being included in a juvenile mental health court can provide psychological, behavioral, educational, social, and familial clinical assessments for use in determining best approaches to treating the underlying causes of many delinquent behaviors. Throughout the 1930s, the Chicago School of Psychology recognized treatment for juvenile offenders that focused on the economic and social aspect of the criminal activities of juveniles, (Granello amp; Hanna, 2003). During this time, juvenile courts were established and designed to yield an alternati ve form punishment of juvenile offenders in an adult criminal system. Emphasis was being placed on rehabilitation instead of punishment; however, in the mid-1980s punishment of juvenile offenders become the top priority. Because of the severe wave of juvenile during the late 1980s, States used the beginning of the 1990s to revise their juvenile statues that they relied so heavily upon. Under new laws, certain charges or offenses required legal responses based on the nature of the offenseShow MoreRelatedHistory of Social Work18530 Words   |  75 Pages..........................................................38 Table: Establishing an Independent Ministry of Social Welfare –Timeline .................................................................39 Subjects allocated to the Ministry of Social Justice Empowerment -India ..............................................................41 Ministry of Women and Child Development -India ............................................. ........................................................42 Subjects allocated

The Shattering of Idealism in Nadine Gerfimer´s The Moment...

Literature changed drastically between the nineteenth-century and twentieth and twenty-first century. Idealistic views that British writers once held, turned into skepticism as Great Britain enter war and inequalities grew greater. The writers of the twentieth and twenty-first centuries wrote realistically what was happening in the world. The Moment before the Gun Went Off by Nadine Gordimer and â€Å"The Day They Burned the Books† by Jean Rhys are both stories that show the shattering of Idealism in twentieth and twenty-first centuries. Britain’s colonialism caused many problems for natives and natural born British who lived in the colonies. The illusion of patriotism shattered as conflicts of race, class, and gender equality took light.†¦show more content†¦One of the many reasons why patriotism was shattered is because coloialism was creating harsh tensions. In The Moment before the Gun Went Off they discuss how the killing of the son is going to turn into an ugly racial debate â€Å"Bad enough to have killed a man, without helping the Party’s, the government’s, the country’s enemies† (p.1442) The reader can conclude from this quote that because great Britain colonized in South Africa it caused rifts between many people. People’s patriotism tends to dwell when all they see is conflict around them. The once idealistic views turn critical when war and inequalities is all there is. The twentieth and twenty-first century writers were common people going through hor rific times such as extreme racism. In The Moment before the Gun Went Off and â€Å"The Day They Burned the Books† both stories leave the reader thinking. Unlike, the 19th century; modern writers often left the end of the story open ended because they didn’t see an end to the conflict. Modern writers often wrote their own personal experiences and feelings on a particular issue leaving much room for the reader to analyze. â€Å"The Day They Burned the Books† tells a story of young boy trying to find where he fits in. His father is from Great Britain and his mother is from the West Indies. Eddie hates England because he associates with his father; a man who is often mean to his mother and says things â€Å"Look at