Recombinant Green Fluorescent Protein Purification

Recombinant Green Fluorescent Protein Purification A series of experiments were performed on the E.coli strain BL21 pLysS pRSETA-GFPUV in order to express and purify a recombinant form of Green Fluorescent Protein (rGFP) using Ni2+-Agarose chromatography based on the rGFP His6 tag properties. A rGFP crude extract (GCE) was collected and later purified resulting in 10 washes and 10 elutions. A Bradford assay was performed on the first 6 samples of the washes and elutions to determine activity via relative fluorescent units (RFUs). A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page) followed to determine purity of the samples and a Western Blot verified the presence of rGFP. The sample with the highest activity was the E3 having 31,927 RFUs with an estimated purity of 10 percent and a molecular weight of 36 kDa. The estimated total yield of our protein is 2.07 ug. Introduction First discovered by Osamu Shimomura in 1961, the Green Fluorescent Protein (GFP) was isolated and identified as a protein that fluoresce green light. When calcium binds to the photoprotein aequorin, in vitro aequorin produces blue light (1). However, in the original jelleyfish A. equorea victoria green light was produced. The green light produced was the result of a second protein GFP driving its excitation energy from aequorin (2). After purification, GFP is a protein of 238 amino acids absorbing blue light at 395 nm and emitting green light at nearly 509 nm (2, 3, 4). This chromophore is very stable towards multiple harsh conditions including extreme pH and heat (5). The Aequorea GFP also measures to be at 30 kDa monomer (6). Purifying rGFP required a unique way of identifying the protein without isolating other protein contaminants. This particular rGFP was tagged genetically to the N-terminal of the protein sequence. This tag is useful due to the unique property of the chromatography technique used. Using Ni2+ -agarose affinity chromatography, the histadine tagged rGFP binds to the Ni2+ , thereby attaching this particular protein to the column. Next, the rGFP is eluted from the Ni2+ -agarose column by running a competitor compound that has a higher affinity for the Ni2+ called imidazole. The His6 tag unbinds from the column allowing the rGFP protein to be collected for experimental purposes (7). The purpose of this experiment is to express and purify the E. coli strain BL21(DE3) using Ni2+ -agarose affinity chromatography followed by the SDS-PAGE and Western Blot procedures to estimate purity and confirmation of the protein. Materials and Methods Expression of rGFP and Preparation of the rGFP Crude Extract (GCE) The BL21(DE3) bacterial culture referred to as G was used to inoculate 10 ml liquid LB [100ug/ml Amp;25ug/ml Cam] growth media and grown overnight at 37 degrees celsius with vigorous shaking. OD600 of .1 of 500 ml of liquid LB growth media was achieved after a second inoculation was achieved with the culture grown overnight. The 500 ml culture was allowed to grow to OD600 reached .5 by vigorous shaking at 37 degrees celsius. The bacterial pellet was stored at -20 degrees celsius and labeled as G0 while 1 ml of the culture was pelleted in a centrifuge tube and induced with IPTG. At the time of induction the cultures relative time is zero. Three hours post induction, the culture was labeled G3, stored at -20 degrees celsius after the supernatant discarded. The same procedure was done to the G3-15 ml with the exception of pelleting 15 ml of the culture. Next, the culture was vortexed at 37 degrees celsius to lyse the bacteria. A 1 ml breaking buffer was added, solution vortexed, and placed in a 37 degrees celsius water bath. After the centrifugation the supernatant was decanted into a new tube labeled GCE representing rGFP crude extract. (8). Purification of rGFP using Ni2+-Agarose Affinity Chromatography A plastic syringe with a luer-lock was blocked by glass wool to hold in the Ni2+ Agarose matrix. The syringe was then secured vertically using a ring stand and filled with approximately 100ul of buffer followed by 2 ml to ensure the removal of air bubbles trapped in the system. A 50 % slurry of Ni2+-agarose was added to 500 ul buffer on top of the glass wool. The system is then opened to ensure packing of the agarose matrix towards the bottom. The final step in setting up the column is the pre-equilibration step which is the addition of breaking buffer to the column while the leur-lock is open until the ethanol is out of the system. After storing away 100 ul of GCE for future use, 1 ml of breaking buffer was added to the centrifuge tube. The GCE was transfered to the Ni2+ -agarose column. After a 10 minute period the luer-lock was opened and .5 ml effluent liquid was collected and labeled W1 followed by W2 until W10. Meanwhile washing the unbound proteins with 4 ml of breaking buffer. After the 10th wash was collected, the column was washed with an additional 5 ml of breaking buffer. A similar process was followed collecting elution 1 through 10 in 1.5 centrifuge tubes, however by adding the elution buffer containing imidazole. The elution buffer formula available in the solution manual (8). Estimating Protein Concentration of rGFP Determine protein amount using the Bradford assay requires a standard curve using known amount of Bovine Serum Albumin (BSA). The Bradford assay was performed on six known amounts of BSA (0, 2.5, 5,10, 15, and 20 ug). 50 ul of solution containing the BSA was added with 1 ml of Bradford reagent to a glass tube. The solution was mixed and incubated at room temperature for 10 minutes. 200 ul of the 6 assays were transferred to a microtiter dish to read the absorbance at 595 nm in a microplate reader. A standard curve was plotted (Absorbance Vs BSA amount) to determine the highest absorbance reading that can be extrapolated from this curve. The same procedure was done on the first six washes and six elutions in triplicate. (9). SDS-PAGE/Coomassie Blue Analysis procedure Two recipes were followed in order to make a resolving gel and a stacking gel. The 12 % resolving gel consists of water, 4x resolving buffer, 30 % Acylamide, 10% ammonium persulfate, and TEMED. This solution was poured between two glass plates until polymerization. The 5 % stacking gel consisted of water, 4x stacking buffer, 30 % Acrylamide, 10 % ammonium persulfate, and TEMED. The Stacking buffer was added on top of the resolving buffer followed by the addition of a toothed comb until polymerization. Afterwards, the samples G0, G3, GCE, W2, W3, E2, E3 were loaded into the gel. The loading of the samples was done after the plates were placed in the electrophoresis tank. The electrophoresis tank then ran for 45 minutes at 200 volts. (10). Preparation and Development of the Western Blot Using the 7 samples from the previous procedure, we add 2-Me (Beta-mercaptoethanol) to each sample and developed another gel. After electrophoresis, the gel was to be used as a part of a sandwich for the Western Blot. The sandwich consisted of (from the top) a clear cassette lid, sponge, filter paper, nitrocellulose, gel, filter paper, and sponge that laid against the black cassette lid. After an incubation period, the protein transferred from the gel to the nitrocellulose. Removing the nitrocellulose to a container, the Ponceau S stain was added for two minutes and rinsed several times with distilled water. This was done until red bands appeared. The molecular weight ladder was marked with pencil for further review. Next a blocking step followed where the nitrocellulose membrane was placed in a Tupperware with 30 ml of 5% non-fat dry milk/TBS solution. The Tupperware was placed on a shaking platform for 30 minutes. Afterwards, the blocking solution was discarded and a washing step compromising of 30ml of .05% Tween 20/TBS solution was added. The Tupperware was placed on a shaking platform for 5 minutes. This process was repeated two more times. Following the washing step a primary probing step was performed by adding 7 ml of mouse IgG anti-Xpress epitope MAb solution and incubated on a shaking platform for 45 minutes. Another washing step followed using 30 ml of .05% Tween 20/TBS and repeated two more times. 7 ml of Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution was added in a secondary probing step that lasted 45 minutes on a shaking platform. The same washing step previous done followed and repeated two times. Finally, the final wash step with 30 ml of TBS was performed on a shaking platform for 5 minutes. For the final step, 7 ml of TMB substrate solution was added to the membrane until band color intensity was achieved. Then the membrane was moved into a Tupperware container filled with water to stop the development. The nitrocellulose membrane was then dried and results recorded. (11). Results The bacterial expression system of rGFP is presented in Figure 1. The Lac repressor is made from Lac1 of the bacterial chromosome. The lac repressor blocks the t7 RNA polymerase but after inducing it with IPTG, the Lac repressor itself will be blocked. Hence, T7 RNA polymerase will start being abundant and be the promoter to the expression for the His6-Xpress-GFPuv thereby resulting in rGFP (7). For a better understanding of rGFP a schematic diagram is provided in Figure 2. The elution of interest was E3 which had 31,927, the highest relative fluorescent units. We also see that from the combined elution profile in Figure 3 which displays the RFU for the first six washes and elutions. The samples were then run through a Bradford assay. The E3 sample had a 20.7 +/- 12.45 ug total amount of protein. The specific activity was 342,995 RFU/mg. The SDS-PAGE gel (Figure 4) presented the molecular weight of E3 standing at 36 kDa. We determined that using the ladder provided. The results gathered was off by 3 kDa from the verified experimental value of the rGFP. From the relative color density, it was determined that the rGFP band retains 10 % purity which resulted in a calculated yeild of 2.07 ug. Figure 5 presents the Western Blot assay which was used to verify the presence of rGFP. E3 and E2 showed a stronger color while G0 as expected did not show a band due to lack of rGFP. The washes also show a faint color. We have confirmed the presence of rGFP by comapring the E3 band to the ladder which approximates 36 kDa. Conclusion/Discussion The confirmation of rGFP was obtained though proceeding with a Western blot analysis. The experiments that were performed beforehand gave a good understanding of how rGFP was induced, expressed, and purified. To recap, Ni2+-agarose affinity chromatography was used to isolate our protein through the unique property having affinity to the His6 tag in the rGFP. Followed by the Bradford assay we estimated how much protein the samples contained. The SDS-PAGe gel gave us an estimation of the molecular weight and purity of the samples which was paramount in the bigger picture of identifying the protein. Even though the purity gained was very low, we proceeded and developed a Western Blot which confirmed the presence of rGFP through band intensities. Since the GFP protein is very robust to pH and temperature, one can transfect or transcribe this gene into other living bacteria or even human cells to possibly see movement. One follow up experiment would to do just that, seeing if we can insert the rGFP into cancer cells or enzymes like insulin for further studies. We already know that GFP changes color based on the excitation energy which might be altered to produce different colors. This can be used to study two systems or their interactions or lack of interaction. We can study the energy consumption of different bacteria and learn which colonies survive longer. We can apply this method in cancer research and find out how cancer fast certain cancers grow by studying the relative fluorescence given off. The impact of this finding may be similar to the impact of creating spectacles (glasses) that allowed millions to see clearly. This protein offers that unique new ability to visually track things we could not have tracked as easily. References O. Shimomura, F. H. Johnson, Y. Saiga. J. Cell. Comp. Physiol. 59, 223 (1962). J. G. Morin and J. W. Hastings, J. Cell Physiol. 77, 313 (1971); H. Morise, O. Shimomura, F. H. Johnson, J. Winant, Biochemistry 13, 2656 (1974). D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. Prendergast, M. J. Cormier, Gene 111, 229 (1992) . W. W. Ward, C. W. Cody, R. C. Hart, M. J. Cormier, Photochem. Photobiol. 31, 611 (1980). Ward, W.W. and Bokman, S.H.: Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21 (1982) 4535-4550. Prendergast, F.G. and Mann, K.G.: Chemical and physical properties of aequorin and the green-fluorescent protein isolated from Aequorea forskalea. Biochemistry 17 (1978) 3448-3453. R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. 84-88 (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. 99-100 (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. 125-126 (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. 139-140 (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. (2012). R. Scott, and E. Picket. Biochemistry Laboratory Manual. United States. (2012). citations: Primary stucture of the aequorea victoria GRP Douglas prasher, virginia eckenrode-229-223 1992 GFP as a marker for gene expression martin chalfie, vuan tu vol 263, feb 1994 Wavelength mutations and post translational autoxidation of GFP vol 91, pp. 12501-1250 dec 1994

Essay --

Approximately three to four percent of babies born every year are born with some kind of genetic disorder. A genetic disorder is described as an illness caused by an error in one’s genome, and is usually hereditary. To understand how these errors occur, one must first understand the basic concept of genes. Genes are the basic units of heredity and are made up of pieces of DNA that instruct the cell how to make specific proteins. Humans are estimated to have about 20,000 to 30,000 genes in their genome. Chromosomes contain these genes and DNA. Humans have 23 pairs of chromosomes or a total of 46 chromosomes. One pair of these chromosomes determines the sex of a person while the other 22 are autosomal, meaning that they determine the rest of the body’s traits, both genetic and phenotypic. Sometimes mutations occur in the genes of the chromosomes which could lead to a genetic disorder or could be perfectly harmless. Other times they happen from the chromosomes themselves i f the chromosome breaks off, switches with another part of a chromosome, or is swapped within the same chromosome. This leads to mutation in gene coding and could potentially cause genetic disorders. There are different types of genetic disorders that each cause different kinds of diseases. Genetic disorders arise from chromosomal abnormalities, single gene defects, multifactorial problems, and teratogenic problems. Chromosomal abnormalities occur when there is an anomaly in chromosome number or structure. The two main types of chromosomal abnormalities are numerical abnormalities and structural abnormalities. Examples of numerical abnormalities are monosomies which is when a chromosome is missing from a chromosome pair and trisomies which is when there is an ext... ...known as balanced translocation is when segments of chromosomes have been exchanged, however nothing is lost or added. A Robertsonian translocation is similar to a reciprocal translocation except that one chromosome attaches to the centromere of another. In an inversion, part of a chromosome has broken off, been turned upside down, and has reattached to itself. Lastly, in a ring, a portion of a chromosome has been broken off and forms circle. Usually these structural abnormalities are due to an accident in one of the gametes, and therefore are also present in all of the body’s cells. However, in some instances, these abnormalities occur after conception, resulting in mosaicism, in which some cells have the abnormality where others do not. In general, chromosomal abnormalities are either inherited or be â€Å"de novo† which means they are new to that specific individual.

Relieving pain and inflammation Essay

1.1 The holistic approach is important as it focuses on relieving pain and inflammation without harming the body. It also promotes the bodies healing response so that the area of injury is healed as quickly and completely as possible. 1.2 One approach to alleviate and minimise pain and discomfort is to show the individual that you are concerned about them and their well being. Let the individual know you are here to help themand want what is best for them. Another approach would be to let the individual know that other people have had the same problem and its nothing to be ashamed or frightened of, some older people may feel they are being a burden and not want to bother anyone, it is important for them to know this isnt the case. Be sincere, smile hold the individuals hand if they are scared. 1.3 Below is an outline of the agreed ways of working to alleviate pain and minimise discomfort. Pain awareness, you should be alert to the possibility of pain and discomfort in older people and that older people are often reluctant to report the pain as they dont want to be a burden to anyone or may be afraid to tell anyone. Pain enquiry, it is impotant to enquire about pain it is helpful if you use alternative words like where are you sore? Have you got an ache anywhere? Are you hurting? Pain description, where pain is present it is important for a clinical assessment to take place. The sensory dimension, the nature (eg sharp, dull, burning), location and intensity of the pain. The affective dimension, the emotional part (eg fear,depression,anxiety) and response to pain. The impact, how is this effecting the individual participation in everyday activities. Pain location, an attempt should be made to locate the pain ask the individual to point out where the pain is. Pain intensity, pain assessment should be made using a numeric scale from 1 to 10 where 10 is the worst pain and 1 being the lowest level of pain, if the individual is able to use this.  Communication, every effort should be made to communicate with individuals with sensory impairments eg glasses, hearing aids etc. Assessment in individuals with impaired cognitive communications (eg dementia suffers) it might be needed for a regular carer and family members to help assess the pain as they will know what is a normal behavior of the individual and one which could suggest the individual is in pain. Cause of pain, a careful physical examination should take place to identify any treatable causes, however it is important to ba aware that pain can exist even if the examination is normal. Re-evaluate, it is important that the treatment is evaluated to make sure it has worked. Read more:  Understand Approaches to Managing Pain and Discomfort 2.1 Severe pain could have the ability to totally transform a person. It could affect the way a person eats and drinks, even putting them off their food completely. pain can cause frustration or unreasonable behaviour in someone who is normally calm and controlled. The individual could becone restless or suffer from sleepless nights all this could be signs of pain and could be used to help diagnois individuals who cant communicate they are in pain. Pain can cause many problems to an individuals well being and communication, pain and discomfort can make day to day life difficult causing limitations to a persons daily activities, this leads to a lower quality of life. Chronic pain causes a host of related problems a main problem can be depression the individual feels helpless and doesnt see a way out. It is important that pain is properly managed so the individual can get their quality of life back and help an individual get back their independance. It is important for none verbal people t o have a way of communication so that they can express their pain, providing the apprropriate pain management assures the dignity and well being of a patient. 2.2 You could encourage an individual to express pain and discomfort in a number of ways the best ways are to make sure the individual feels safe and at ease with you, smile sincerely, ask the individual an open ended question, be supportive make sure the individual knows they are not alone, let them take their time to open up to you. 2.3 There are a number of self-help methods of pain control it is important to make the individual aware of so they can use them. Some are more successful then others. The individual could be encouraged to do some gentle exercise or have some physiotherapy where possible. There is a distraction approach where individuals are incouraged to do something they enjoy such as reading, drawing, watching television or maybe listening to music to take there mind off their pain. Sometimes a lie down or rest can help relieve pain maybe the individual could be incourage to take a rest when the pain is really bad. It can sometimes help if the individual has a bath or shower the warmness of the water can ease pain in some cases. 2.4 Assist an individual to be positioned safely and comfortably, this may be different for different individuals it is important to consult the individuals care plan. Once an individual is moved according to their care plan and in compliance with safe moving and handling guidelines you may need to make them more comfortable using aids. The individual may need more cushions to make them more comfortable, they may need their feet elevating, the person may need to be seating in a recliner for comfort. 2.5 Measurements to minimise the individuals pain and discomfortmay include repostioning, adjustments to bedding, heating, lightening or noise, the use of specialist mattresses and pressure reducing aids, Also analgesias Pain killers) maybe requested for the individual all measures in the care plan must be followed. The ways pain and discomfort may be managed include massage, yoga, meditation and medication. 3.1 Carry out all monitoring according to the individuals care plan. The individual maybe on 15 min, 30 min, or hourly checks these need to be carried out. If the individual can communicate it is important for you to ask them about any pain, has the individuals mood or behaviour changed are the signs of pain and discomfort there. 3.2 Records should be completed in a required way as explained in the individuals care plan. All pain killers administered should be documented on the MARS sheet. 3.3 All findings and concerns should be reported correctly to the person in charge and documented in the care plan.

How to Negotiate Your Salary in an Interview

How to Negotiate Your Salary in an Interview In negotiations, the first person to blink usually loses. The same goes for salary negotiations. If you name a number first, you’ll never know how high the hiring manager might have gone to win you. Here are five sneaky ways an interviewer will get you to answer the money question, and how to avoid them.Q: â€Å"What is the salary range you’re expecting?†Your ideal answer: â€Å"I’d like to get a better sense of the requirements before I commit to a number. Just so I can make sure I have a sense of what you need.†Q: â€Å"How much did you make at your last job?†Your ideal answer:  Don’t answer it. Say instead: â€Å"First I want to make sure I understand the ways in which this position’s responsibilities will differ from those of my former position. Let’s discuss the details before we agree on a fair amount.†Q: â€Å"What are you hoping for in terms of salary?†Your ideal answer:  This is basically the sa me as the first question. If they’ve already asked some version of this, try this answer, and keep deflecting: â€Å"I’m sure whatever you’re offering will be commensurate with the going market rate for this position.† This puts the burden of fairness on them.Q: â€Å"In order to make you an offer, I’ll need to know your requirements.†Your ideal answer:  False! Resist! Deflect again! How about: â€Å"Let’s start with what you have budgeted for this position and then we can discuss from there.†Q: â€Å"Why don’t you want to disclose your salary requirements?†Your ideal answer:  This is quite the bold one, and not all that common. At this point, it’s okay to fight fire with fire. Try: â€Å"I’d really like to get a sense of what this position is worth to your company before I make any commitments.†As tough as it is to be tough, it will pay out in the long run. You may feel awkward about taki ng such a hard line, but your interviewer will respect you as someone not to be trifled with. You might even win yourself the offer with your negotiating prowess.

The Reconstruction of Eurpoe After World War II essays

The Reconstruction of Eurpoe After World War II essays THE RECONSTRUCTION OF EUROPE AFTER WORLD WAR II Even while World War II war still being fought, leaders of the Allied powers began thinking about how to reconstruct Europe after the war. Winston Churchill of Britain, Franklin Roosevelt of the United States, and Joseph Stalin of the Soviet Union agreed first of all, that they would need to root out fascism entirely. This meant they would not only need to defeat Germany but also need to denazify Germany by punishing those who were responsible. Coming out of the Second World War which completely ruined and crippled Europe, many countries had to face the problems of material, economic and moral reconstruction. This report shows how the countries went about rebuilding a ruined Europe. On June 6th 1947 George c Marshall presented a speech of a plan the forever-changed Europe. It was a plan to rebuild the destructed Europe after World War II. ?Borne from the mind of a wise and diplomatically skilled visionary, the Marshall Plan was the phoenix on whose wings war ravaged Europe would begin its ascent from the ashes of World War II. The Plan took root in the Economic Cooperation Administration (ECA) created by Congress in April 1948. Its official title was the European Recovery Program. It is called the Marshall Plan, however, in honor of its creator - Secretary of State George C. Marshall. Constituting one of our nation's finest for eign policy moments, the Marshall Plan signaled America's unequivocal resolve to assist an economically struggling Europe, and assume a position of leadership on the post-WWII stage. Observing the financial crises which had forced Britain to pull out of Greece, the massive European capital shortages, poor crop conditions, rising inflation, and the budding seeds of communist parties in France and Italy, Secretary of State Marshall was determined not to repeat the mistakes of World War I by simply standing by as bad times turned worse. At the Harvard U...

Global strategy (MBA market) Essay Example | Topics and Well Written Essays - 2250 words

Global strategy (MBA market) - Essay Example In seeking to establish a Business School in London, England, this business plan will build a sales, investment, marketing, and operations plan for the ‘International School of Business Innovation’ to be established in 2011. The school will initially pursue a goal of enrolling domestic and international students in an online program with a 4 week on-site seminar in London conducted yearly. In initiating operations, the school will market to and see the enrollment of both foreign and domestic students. Due to the limitations of budget, initial marketing internationally will be conducted in a manner that targets the students of India and Pakistan particularly, due to the demand for higher business education in those countries popularly. PART 2: BUSINESS PLAN: 1. Introduction In building and establishing a Business School in London with an intention of offering MBA (Masters of Business Administration) degrees to international and domestic students, the most important factor s are the business plan, philosophy of education, and location. In searching for the ethos for the school, the general approach to business that would guide operations from a position of philosophy, the management committee evaluated the writing, work, and experience of many of the top 100 CEOs internationally and historically. One of the most respected CEOs is Jack Welch, the former General Electric business leader who is well known for innovation in the corporate sphere and managing companies at the highest level. Jack Welch is currently associated with an online MBA school in the United States that offers an affordable business education to students anywhere in the world. â€Å"As a part of his effort to provide quality educations at an affordable cost, the Jack Welch Management Institute moves away from the stereo type business institutions that charge their students an average $100,000 for a MBA degree . On average, The Jack Welch Management Institute charges $600 per credit h our. This translates to students receiving an accredited MBA degree for just over $20,000. The realization of his dream for a management institute came through the coming together of a group of investors led by Michael Clifford who purchased the ailing Myers University in Cleveland in 2008. These investors hammered out a deal with Welch to establish the Jack Welch Institute of Management based on his management style and philosophy that brought him and general electric to the heights they both attained in society.† (OnlineEdu, 2010) As a start-up business venture, the MBA School proposed for establishment in London will follow the online MBA program model given as example in the Jack Welch Management Institute and seek to provide affordable, world-class business education to students worldwide in the form of an online-only school. 2. Overview of International Business School The first aspect required for the development of the project of creating an online MBA program for inte rnational and domestic students is to create a management team and business plan for the venture. In managing the main operations executively myself, I see also a need for a minimum of a five person management team who would oversee the establishment of the school. This group would lead the effort to secure financing, sign the lease for the

PBL2 Essay Example | Topics and Well Written Essays - 1500 words

PBL2 - Essay Example The ischemic necrosis of kidneys gradually heals by undergoing progressive fibrous scarring (Alpers, pg. 1012). Destruction of extracellular matrix occurs. The regenerative capacity of renal tissue is maximal in cortical tubules, less in medullary tubules, and nonexistent in glomeruli. Hence this is the correct answer. B: Granuloma formation: Granulomas are formed following chronic inflammation and are encountered in some immunological mediated reactions, infections and some non-infections conditions. Some of the common conditions in which granulomatous inflammation occurs are tuberculosis, sarcoidosis, cat-scratch disease, leprosy and syphilis. The granuloma consists macrophages mainly (Kumar, pg.83-84). Hence this is not the correct answer. C: Liquefaction: Liquefaction or liquefactive necrosis is a condition in which the affected cell is completely digested due to powerful hydrolytic enzymes. It usually occurs in fungal and bacterial infections and causes formation of abscess. Also, ischemic injury in brain causes liquefaction (Mitchell, 138). Hence this is not the correct answer. D: Metastatic calcification: Deposition of calcium salts in otherwise normal tissue is known as metastatic calcification. It occurs due to elevated calcium levels. It is commonly seen in the kidneys and lungs. Hence this is not the correct answer. A 25 year old-woman sustains a deep laceration over the right forearm in a motorcycle accident. The wound is cleaned and sutured. During the next 3 months, the wound heals with formation of a linear scar. Which of the following nutritional factors is required for proper collagen assembly in the scar tissue of the patient? A. Folic acid: Folate, the useful form of folic acid is an essential nutrient for the production and maintenance of new cells because it is needed for the replication of DNA. It is not useful for collagen production. Hence this is not the correct