Induction of Aminolevulinic Acid Synthase Gene Expression

Induction of Aminolevulinic Acid Synthase Gene Expression Induction of aminolevulinic acid synthase gene expression, down-regulation ferrochelatase and enhancement of metabolite, protoporphyrin IX, excretion by co-therapy with isoniazid and rifampicin (1. Isoniazid and rifampicin induced liver injury by regulating 5-aminolevulinate synthase and ferrochelatase and enhancing protoporphyrin IX 2. Mechanism of rifampicin and isoniazid induced cell death in L-02 cell line and mice) Abstract Isoniazid(INH) and rifampicin(RFP) are first-line antituberculosis drugs, co-therapy with INH and RFP is highly effective. However, the combination of these two drugs frequently cause liver injury or liver failure in humans. The risk of hepatotoxicity is considerably higher in patients receiving both RFP and INH than in those receiving either RFP or INH alone. Numerous studies have been conducted to investigate the mechanism of injury after isoniazid or rifampicin used in various animal models, however, the important mechanism for the combination of isoniazid and rifampicin in humans remains unclear. Here we investigated this combination induced hepatotoxicity using L-02 cells and mice. Introduction Tuberculosis remains a global public health problem whose effects have major impact in developing countries. World Health Organization estimates that there were 8.6 million new TB cases in 2012 and 1.3 million TB deaths. The currently recommended treatment for new cases of drug-susceptible TB is a six-month regimen of four first-line drugs: isoniazid, rifampicin, ethambutol and pyrazinamide. (Global tuberculosis report 2013). However, the combination of isoniazid(INH) and rifampicin(RFP) frequently cause liver injury or liver failure. The risk of hepatotoxicity is considerably higher in patients receiving the combination than in those receiving either RFP or INH alone. The mechanisms leading to liver failure in humans were poorly understood. Recently, a new mechanism ,independent of INH metabolism, is found in the RFP and INH co-therapy induced liver injury. Li et al. (Li, et al. 2013) found that co-therapy with RFP and INH targets porphyrin biosynthesis and results in hepatic protoporphyrin IX (PPIX) accumulation and liver injury . PPIX is an intermediate in porphyrin biasynthesis. Normally the concentrations of PPIX is very low in the liver. However, in some cases the concentration abnormally elevated in blood and liver, such as erythropoietic protoporphyria. High concentrations of PPIX in the liver are known to cause liver injury (Anstey and Hift 2007; Casanova-Gonzalez, et al. 2010). Using hPXR mice, Li et al. demonstrated that the accumulation of endogenous PPIX is through PXR-mediated transcriptional activations of aminolevulinic synthase-1(ALAS1) genes. ALAS1 is the rate-limiting enzyme of heme synthesis in the liver and is drug-responsive, providing heme for CYPs and other hemoproteinsis. Activation of PXR can up regulate ALAS1 expression in liver (Fraser, et al. 2003). RFP upregulate ALAs1 increasing heme-biosynthesis in the liver and overproducing PPIX through activating PXR signalling pathway. However, PPIX accumulation strongly suggests that ferrochelatase became a ratelimiting enzyme during INH-RFP treatment (Lyoumi, et al. 2013). Ferrochelatase (FECH) ,the final enzyme in the heme biosynthetic pathway, catalyses ferrous iron inserted into precursor porphyrin protoporphyrin IX to form heme, and when defective or deficient, causing accumulation of protoporphyrin IX. Ferrochelatase is active in cells that produce 80% heme in the bone marrow (Bloomer, et al. 1991) and the rest in hepatocytes (Bonkowsky, et al. 1975). The excess protoporphyrinIX becomes insoluble in bile and exerts cholestatic effects leading to architectural changes in the hepatobiliary system ranging from mild inflammation to fibrosis and cirrhosis (Anstey and Hift 2007). MATERIALS AND METHODS PI staining L-02 were allowed to adhere on glass bottom dishs for 4h, followed by INH,RFP or INH/RFP. The medium was removed after h and cells were stained with for 30 min. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and images were recorded with a fluorescence microscope. Western blotting L-02 cells cultured in flask were harvested using 0.25% trypsin (Hyclone, Thermo Scientific, Waltham, Mass). After centrifugation at 1000r and lysis using buffer for Western blotting (), total proteins were collected by following the kit instructions. Protein concentrations were determined using the BCA Protein Assay Kit (). After heating at 95 °C for 5 minutes in sample buffer, proteins were separated on SDS-PAGE using 10% polyacrylamide gels before electroblotting onto PVDFmembrane(). Nonspecific binding was blocked by incubation for 2 hours in 5% (w/v) nonfat milk. The following primary antibodies were used overnight at 4 °C: Rabbit anti-human FECH antibody(; 1:1000); Rabbit anti-human ALAs1 antibody( 1:500); Rabbit anti-human BCRP antibody(; 1:500). Bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Beijing Biosynthesis Biotechnology Co. LTD). Finally, the membranes were visualized by chemiluminescence. RNA Isolation and Real time Polymerase Chain Reaction for ALAs1 and FECH Cell Culture L-02 cells, a human fetal hepatocyte line, purchased from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, purchased from Shanghai, China, were cultured according to the manufacturer’s instructions 15 at 37 °C in 5% CO2. Cell culture materials were procured from Corning() Discussion Nevertheless, the ability of chemicals to activate PXR is species dependent. RFP is a human PXR specific activator that weakly affect on mouse (Lehmann, et al. 1998). INH hepatotoxicity is thought to be dependent on metabolic activation by arylamine N-acetyltransferase and CYP2E1, but Li found AcHZ and hydrazine do not cause INH-related hepatotoxicity. Hepatic heme synthesis leading to protoporphyria and possible impact with other metabolic systems (Davies, et al. 2005). References Primary Sources Secondary Sources Uncategorized References Anstey, A. V., and R. J. Hift,  2007, Liver disease in erythropoietic protoporphyria: insights and implications for management. Postgrad Med J 83(986):739-48. Bloomer, J. R., et al.  1991, Heme synthesis in protoporphyria. Curr Probl Dermatol 20:135-47. Bonkowsky, H. L., et al. 1975, Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts. J Clin Invest 56(5):1139-48. Casanova-Gonzalez, M. J., et al.  2010, Liver disease and erythropoietic protoporphyria: a concise review. World J Gastroenterol 16(36):4526-31. Davies, R., et al.  2005, Hepatic gene expression in protoporphyic Fech mice is associated with cholestatic injury but not a marked depletion of the heme regulatory pool. Am J Pathol 166(4):1041-53. Fraser, D. J., A. Zumsteg, and U. A. Meyer,  2003, Nuclear receptors constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene. J Biol Chem 278(41):39392-401. Lehmann, J. M., et al.  1998, The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102(5):1016-23. Li, F., et al.  2013, Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid co-therapy. Nat Med 19(4):418-20. Lyoumi, S., et al.  2013, PXR-ALAS1: a key regulatory pathway in liver toxicity induced by isoniazid-rifampicin antituberculosis treatment. Clin Res Hepatol Gastroenterol 37(5):439-41.

The Many Faces of Freedom? Essay -- Expository Exemplification Essays

The Many Face of Freedom? Freedom is a concept that people are often willing to die for and it is the cause of much fighting. However, few people ever claim to dislike freedom. This raises an interesting question: how can people fight over what is generally considered to be a positive idea? Does this mean that someone must be against freedom? The answer is that people cannot agree on what freedom is, thus numerous groups can claim to be "for freedom" while strongly disagreeing on the means by which to achieve it. These groups often argue vehemently and passionately, trying to convince the majority that their side is right. However, emotion is only one part of deciding who is more persuasive. I offer two examples of disagreements regarding freedom, as proof that freedom is neither tangible, nor a singular idea. An example of a disagreement about freedom between two larger groups is offered in Michael Rossman's account of a student protest in "The Wedding Within the War". Feelings between students and the administration came to a head in an argument regarding tables set up by student organizations to meet new members and pass out information. The administration first restricted the students' rights by forcing them to move the tables from the heart of campus to the edge of campus, further from the majority of students. Then, a few years later, the students were told that they were not allowed to have the tables at all (102). Since their campus is a microcosm of the larger government of America, this limiting of their rights frightened them, causing them to react. As a result, they held a demonstration to make these concerns heard. Their main point, as presented in "Catch-801" by Marvin Garson was that "the University Administ... ...s to be a singular concept. The personal quality of an individual's definition of freedom is also the reason why the students were able to be more persuasive. Their writing contained a sense of personal concern, that decisions made regarding freedom would impact each one of them individually. In contrast, political speeches, although concerned more with the majority, spoke more in terms of abstract freedom, which is much less persuasive. Works Cited Garson, Marvin. "Catch-801." Takin' It to the Streets. New York. NY: Oxford University Press, 1995. Reagan, Ronald. "Freedom vs. Anarchy On Campus." Takin' It to the Streets. New York. NY: Oxford University Press, 1995. Rossman, Michael. "The Wedding Within the War." Takin' It to the Streets. New York. NY: Oxford University Press, 1995. Roth, Philip. Goodbye, Columbus. New York. NY: Bantam Books, 1968.

Holding Parents Accountable for Their Childrens Behavior :: essays research papers

In the past, there have been many minors who have done numerous of acts in which they are punished in a reasonable manner. Just imagine if the parents of these children were put on trail instead of the minors. Why should a parent have to suffer the consequences for their child’s mistake, in which they probably had no idea what the child was doing.   Ã‚  Ã‚  Ã‚  Ã‚  According to The Beaufort Gazette in Beaufort, SC, â€Å"A couple in Boise, Idaho now faces criminal charges because their 10- and 12-year old sons sexually molested three of their younger siblings, starting when one of the children was just one month old.† The report goes on to say, â€Å"Prosecutors said one girl was just 2 years old when the abuse started in October 2002. The other girl was less than 8 months old. The sexual abuse of the boy allegedly began in December 2002, when he was just one month old.†   Ã‚  Ã‚  Ã‚  Ã‚  According to The Idaho Statesman Newspaper in Boise, Idaho, â€Å"The parents are being charged with four counts of felonies. The first three include the connection with the allegations of sexual abuse by the boys, and the fourth charge addresses allowing a child to walk on dirty floors, in which resulted in infected cuts on the child’s feet.†   Ã‚  Ã‚  Ã‚  Ã‚  This sounds more like a child neglect case rather than a connection of a child molestation case. I am not defending the couple in any way, but I feel there is no proof of them knowing about such acts going on between their children. Therefore, it will be hard to prosecute the couple on the molestation charges unless there is some type of evidence that is not being presented or considered until the trail.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  There was a

Film and Television Criticisms: Similarities and Differences of Male Characters Essay

Male portrayals on televisions have greatly evolved from the standard hero stereotypical illustration of primary male characters. During the entry of 21st century, masculinity among male roles have expanded and included variety of multicultural forms. In fact, the current illustration of masculinity has adapted to the liberal trends of society and even considered various facets of sexual considerations than the common romanticized absolute hero-portrayal of males. In addition, the male sexual domination against feminism is now becoming less frequent compared to the males roles of the 20th to early 21st century. Background of the Problem During the entrance of 21st century, various cultural modifications in various countries worldwide have occurred due to globalization, western influences and the decline of the concept on gender discrimination. Due to the vast liberalization on gender and influences of globalize and liberalized environment, communities, especially in United States, are now evolving the gender portrayals from conventional to a more equalized stature. As for these socio-cultural modifications, gender roles in media and entertainment are also being influenced by the shifting of idealism. In terms of male roles in entertainment, the common hero stereotypic roles implicating absolute masculinity have already diverted in accordance to the prevailing trends of the society. Since the public is now open to different variations of sexualities, male behavioral patterns and the society’s awareness on multi-gender variations, roles portrayed by males in variety of television programs have been modified to more multi-faceted and diverse forms. In an effort to illustrate these conditions, three movies with different genre and cultural implications have been selected are analyzed based on the similarities and differences of male roles in the television programs of the 21st century. Discussion The Male Characters of Prison Break Prison Break (2005) is an action, thriller and drama- based television series created by Paul Scheuring with the primary characters, Michael Scofield (portrayed by Wentworth Miller) and Lincoln Burrows (portrayed by Dominic Purcell). The two main protagonists play the role of brothers who aimed at escaping the prison penalty before Lincoln faces his trial of execution penalty due to the false accusation of murdering the vice-president, Terence Steadman. The two characters are hindered by the antagonist group called, The Company; although, despite of the trials and obstacles beneath the prison walls of Fox River State Penitentiary, the brothers are able to save themselves from the grasps of the covert agents of the said group. Prison Break is one of the best male role portrayals in the television series as of 21st century. From the given overview, the brothers are confronted by the issues of their past and the accusations made against Scofield’s brother, Lincoln. Three of the most notable strengths of the brothers are (1) brotherly bond to each other, (2) Scofield’s expertise in construction engineering and Lincoln’s genius skills, and (3) their comrades who are also escapees of the prison. Meanwhile, most weaknesses observed among the brothers are sometimes (1) their immediate concern to their comrades ending to self-risks, and (2) frequent conflicting plans and misunderstandings. Despite of these strengths and weaknesses, the brothers are motivated to escape the prison and live the free status that they are supposed to possess. Schofield already considers the little or zero possibility of uplifting the verdict to his brother; hence, both of them hope to escape the grasps of their chasers and live out of the claws of their enemies. The roles of the two characters are portrayed in a masculine sense where the sensitive attachments as brothers are very much evident. From the physical features and role portrayed by the brothers, each role manifests a sense of interdependency with each other. The Male Characters of Dante’s Cove Dante’s Cove (2005) is another film oriented to a horror and LGBT (Lesbian, Gay, Bisexual and Transgender) genres created by Michael Costanza with gay couple, Gregory Michael and Charlie David, as the primary characters of the series. The television program is another face of the male role portrayals that are liberally being introduced to the public in the 21st century. The gay sexual orientation of males is one of the considerations to the vastly evolving male culturalism in the film industry, which essentially provide a different course depiction of male portrayal. In the story, Kevin (portrayed by Gregory Michael) is a formerly discreet bisexual who happened to fall in love with his seasonal buddy, Toby (Charlie David) who actually works as a bartender in a haunted hotel at Dante’s Cove. During the middle section of the first season, Kevin is able to realize what he wants and decides to leave his prosperous life with his mother and discriminating father-in-law to live in with Toby at Dante’s Cove. Upon Kevin’s arrival to the area, he is confronted by various premonitions from the warlock antagonists, Ambrosius Vallin (portrayed by William Gregory Lee). In the story, Kevin has accidentally freed the warlock by a simple kiss from the prison-enchantments of another antagonizing character, Grace Neville (portrayed by Tracy Scoggins) – the witch of Dante’s cove. By freeing Ambrosius from his prison, he sets out to hunt his so-called destined lover in the persona of Kevin. Meanwhile, being the former lover of Ambrosius, Grace hunts the love of Ambrosius (Kevin) in order to avenge herself from the warlock’s betrayal of her love 50 years ago. The couple is now confronted by the immense witchcraft of the two members of high-orders. The task of the Kevin and Toby is to maintain their emotions to each other despite of the efforts of the two antagonists in breaking their relationship apart. From the given overview, the strengths of the primary characters present in the film are the (1) emotional bonds to each other despite of gender issues and (2) the aid coming from their comrades from the cove. However, certain weaknesses observed in their male portrayal are (1) their fragility against lies, (2) gays’ stereotypes of polygamous nature, and (3) the emotional set backs that occur between the two. Meanwhile, some of the observed motivational behaviors present in the two primary characters are their attachments to one another and the supporting atmosphere they obtain from their bisexual and lesbian friends. Throughout the film, Toby and Kevin hope to finally obtain peace with their ideal form of gay relationship; however, the greatest fear confronting the two is their separation from one another. Despite of their gender similarities and the moral-culturally considered taboo, the couple has evidently established their ideal perspective of gay relationship while maintaining the external nature of their masculine behaviors. The Male Characters of Heroes Heroes (September 25, 2006) is a drama, science fiction television series created by Tim Kring with his primary character, Peter Petrelli (portrayed by Milo Ventimiglia). The story revolves in the discreet existence of evolved human beings capable of using unnatural powers inherent within their genetic structures. Each evolved superhuman possess either destructive or supportive form of unique abilities. The main antagonist, Sylar (portrayed by Zachary Quinto), is an evolved form capable of absorbing the powers of other superhumans by actually devouring their brains. Sylar moves with his intent of capturing the key to his immortality with the power of the Cheerleader, Claire Bennet (portrayed by Hayden Panettiere). Unlike Sylar, Peter Petrelli possesses the unique ability of absorbing one’s power by simply getting near towards these people. Unfortunately, he enters in without recognition in his skill and unable to manipulate the absorbed powers at his will. Peter is confronted by the complex task of saving his kind from the deadly virus released by a group of individuals who wants their kind annihilated. The science fiction film revolves in the lives of various complicating lives of different characters of the film; however, the concentration of tasks and the primary role as the hero is vested in the character of Peter Petrelli. In terms of his strengths as the male role of the film, he possesses (1) distinct and non-replicable skill of obtaining one’s ability in the simplest way, and (2) comrades that are also equipped with unique abilities. Meanwhile, despite of the heroic character of Peter, his identity in the film is surrounded by critical weaknesses that serve as his primary obstacles prior to achieving his goal of defeating his antagonists. Some of these identified weaknesses are (1) his incapacity to control his powers and abilities at his will, (2) his fragile emotions when it comes to his brother Nathan Petrelli and his loved ones, (3) unable to recognize his own potential, and (4) his fear towards his own abilities. Meanwhile, despite of the weaknesses of his character, Peter is motivated by lost of his brother, Nathan, the death of his loved ones, and the abduction of his girl during his travel in the future. Out of these obstacles and discouragements, Peter still hopes to rescue his girl and his brother from their circumstances; however, he is still confronted by the fear of the destruction he can cause and fear of loosing his love ones in his own hands. Conclusion: Analysis of the Three Chosen T. V Programs In analysis of the male character portrayals from the three chosen television programs, particularly Prison Break, Dante’s Cove and Heroes, there are certain similarities and differences observed among the characters of the said programs. In consideration of similarities, Prison Break’s brothers – Michael Scofield and Lincoln Burrows – depict the anti-heroic roles in terms of the storyline’s plot. The masculine sides of the brothers are further exemplified by their complex relationships involving a different sense of heroic act compared to the conventional heroic roles of male portrayals in the past. Meanwhile, Dante’s cove similarities in Prison Break is its ironic male roles of anti-masculine portrayal in terms of sexual orientations in the film wherein Kevin and Toby are confronted by the issues of saving their queer relationship being confronted by the horrifying witchcraft of the antagonists. Lastly, Peter Petrelli of Heroes is more similar to Prison Break’s brotherly linkage as with his brother, Nathan Petrelli, who is very much emphasized in the plot of Peter’s journey. In terms of the character similarities, the primary male roles of the said three television programs have already diverted to a different heroic stereotype common in the 20th century film plots. Meanwhile, in terms of the differences showed by the three primary male roles, each possesses differences in relation to the use of multi-culturalism components, gender portrayals, and defining characters of masculinity. As for Prison Break, the brothers are confronted by the emerging complexities of brotherly conditions. In a cultural sense, Prison Break illustrates the conventional role of brotherly affection; however, gender concerns might suggest the questionable bonds of brothers. In addition to cultural components, the nature of their role as prison breakers even distort the 20th century heroic male roles, such as the romanticism influenced-heroes. In Dante’s Cove, the male roles of Kevin and Toby are confronted by issues of cultural liberalization in terms of their illustration of free manly affection, which is actually considered non-manly by cultural norms. However, as for the film and the definition of masculinity, Dante’s Cove is able to raise the concept of manliness in a more behavioral sense than with sexual choices or preferences. Obviously different from the two male portrayals of Prison Break’s brothers and Heroes Peter Petrelli, Dante’s Cove couples have altered the components off masculinity by portraying it outside the common stereotypes of male film roles. Lastly, the character of Peter Petrelli in his diverse heroic role in Heroes has actually portrayed a fragile heroic role. Initially with his low self-compliance and belief in his capacities, his masculinity is confronted by a weaker illustration of identity, which is another diversion from the usual romanticism heroic view. In conclusion, as of 21st century, male roles in television programs have indeed evolved to more complex and diverse heroic portrayals.

Is It Better for People to Stop Trying When They Feel Certain They Will Not Succeed?

Assignment: Is it better for people to stop trying when they feel certain they will not succeed? Although there is the notion held by some people that we should give up some tasks in our life which â€Å"seem† not to be successful forever, I really consider that we should insist on everything we want to do. For, the consistency and the durability of the certain thing will lead us to splendid success. Due to Continuation, Mathew Emmons, a famous sport shooter, and Alfred Nobel, known for his invention of dynamite, earn themselves irreplaceable fame in their respective fields.Matthew Emmons, though missed the Olympics Games champ in shooting all the time, still impresses us with his fortitude. As a famous American shooter, Matthew attended Olympics Games for three times separately during the past twelve years; he missed the first place unchangeably. Nevertheless, he always appeared at the great games, chasing after his dream to become the champ in shooting. During the Olympics o f Athens, he let his champ go by bombing in the last shoot. Matthew, though frustrated, still collected enough courage to participate in the following games in Beijing and London.Unfortunately, misfortune happened on him again; He implausibly scored only few points in the last shoot in the final of his games, narrowly escaping the champ. Afterwards, interviewed by journalists, Matthew said that he would assiduously prepare for Rio de Janeiro. Undeniably, he might feel distressed when he failed in so many great games; he never gives up. Instead, he insists on training and hopes to attend the Game in Rio de Janeiro, 2016. At the same time, the unstopped experiment made by Alfred Nobel can illustrate my idea.Alfred Nobel owes his irreplaceable fame as an inventor of dynamite. Alfred Nobel cultivated great interest in science, especially in explosive, since very young under the influence of his father. Alfred Nobel engaged himself into experiments to find a more powerful and stable subs tance after his graduation from the college. The procedure proved to be tough, even frustrated. On 3 September 1864 a shed, used for the preparation of nitroglycerin, exploded at his factory, killing 5 people including Nobel's brother.Nobel was not obsessed with the bitterness of such accident; instead, he became more strengthened about his goal to find new chemical substance to replace nitroglycerin. Nobel was eventually rewarded when he successfully invented dynamite in 1867. We had to admit that Nobel suffered a lot during the experiment: lose his brother, lose many friends, and lose the trust from the family. However, he had no idea to give up the research on the Dynamite. Through our constant efforts, we can best demonstrate our ability and pursue our dreams. Maybe, the result may change miraculously towards direction desired by us.

Protein Characterization by Electrophoresis

EXPERIMENT NO. 15 PROTEIN CHARACTERIZATION BY ELECTROPHORESIS Abstract The molecular weights of protein extracts were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two sets of four protein samples, standard bovine serum albumin (BSA), invertase, egg albumin, and casein, were prepared; one set containing ? -mercaptoethanol (BME) while the other did not. These were then analyzed through SDS-PAGE with 12. 5% resolving gel, prepared using 2 M Tris-HCl at pH 8. 8 and stacking gel, prepared using 0. 0625 M Tris-HCl at pH 6. . Results showed multiple bands located on the upper half of the gel, which suggested heterogeneity of the mixture and that the samples were heavy molecules. Introduction Proteins are biological macromolecules composed of one or more polypeptides, which are polymers of amino acids. Structurally diverse, these molecules also serve a myriad of functions from enzymes, which are the biological catalysts of many physiological reactions, to components that maintain the structural integrity and organization of cells (Pratt and Cornelly, 2011). Because of this, it has been a constant effort among chemists to extract and isolate proteins to determine the mechanisms by which they act and produce the results of their reactions. Further knowledge of their biological action could translate into the discovery of many resources that could facilitate humans’ and other species’ daily lives. Electrophoresis is an analytical tool through which one can examine the movement of charged molecules in an electric field. Many modern electrophoretic techniques use a polymerized gel-like matrix as a support medium. The molecules’ migration is dependent on the applied electric field, the rigid, mazelike matrix of the gel support, and their size, shape, charge, and chemical composition. The movement of a charged molecule in an electric field is given by: v=Eq? f (1) where v is the velocity of the molecule, E is the electric field magnitude, q is the net charge of the molecule, and f is a frictional coefficient dependent on mass and shape of the molecule. Hence, it is observed that under a constant electric field magnitude, the movement is dependent on the charge-to-mass ratio of the molecule. Since each molecule is expected to have unique charges and sizes, their mobility under the electric field would also be different. Gels used in electrophoresis with different pore size may be produced by using different concentrations of cross-linking agents. Polyacrylamide gel electrophoresis (PAGE) allows enhanced resolution of sample components due to separation based on molecular sieving and electrophoretic mobility. Because of the presence of a continuous network of pores in the gel, large molecules do not move easily through the medium compared to smaller ones. Two types of gels are used: the resolving and stacking gels, each having different concentrations of acrylamide and of different pH and ionic strengths. The denaturants sodium dodecyl sulfate (SDS), a detergent, and ? -mercaptoethanol (BME), a reducing agent, are frequently used in PAGE. The action of these two denaturating agents cause the production of polypeptide chains of constant charge-to-mass ratios and uniform shapes due to the SDS molecules binding with the hydrophobic regions of the denatured polypeptide and masking the native charge of the protein by its negative charge. This restriction, coupled with the fact that mobility of the SDS-protein complexes are based on molecular size, forms the basis of the electrophoretic determination of purity and molecular weight (Boyer, 1993). This experiment will utilize SDS-PAGE to assess the molecular weights of the extracted proteins invertase, albumin, and casein, along with standard bovine serum albumin. The effect of the presence of ? -mercaptoethanol was also investigated. Methodology With the glass plates clean, the gel apparatus was first set up with the comb inserted between the glass plates. It was made sure of that the set-up would not leak by allowing a little amount of distilled water to enter it, which was discarded afterwards. A mark, one centimeter below the teeth of the comb, was placed on the glass plate. The resolving gel, at 12. 5% gel, was then prepared in an Erlenmeyer flask. Using a micropipette, 1450 ? L of 40% stock acrylamide, 775 ? L of 2% stock bisacrylamide, 875 ? L of 2 M Tris-HCl at pH 8. 8, and 1500 ? L of distilled water were measured and mixed in the Erlenmeyer flask. Afterwards, 47 ? L of 10% sodium dodecyl sulfate (SDS) and 40 ? L of freshly prepared 10% ammonium persulfate (APS) were added to the mixture. Then, ten microliters (10 ? L) of tetramethylethylenediamine (TEMED) was added and, after mixing it by swirling not more than three times, the mixture was poured into the gel apparatus with the aid of a micropipette up to the mark. The gel was then overlaid with a small amount of isobutanol-water mixture before it would start to harden. After the gel has completely polymerized, the isobutanol mixture was removed from the apparatus. Two pairs of two resolving gels were prepared as one pair would be used for samples containing ? mercaptoethanol and another pair for those that do not contain the said chemical. The stacking gel was prepared by taking 265 ? L of 40% stock acrylamide, 140 ? L of 2% stock bisacrylamide, 350 ? L of 0. 625 M Tris-HCl at pH 6. 8, and 940 ? L of distilled water and mixing all four in an Erlenmeyer flask. Afterwards, 25 ? L of 10% SDS and 60 ? L of 10% APS were added to the one in the flask. Immediately before the solution was added, 5 ? L of TEMED was added to it and swirled not more than three times, similar to that done with the resolving gel. This mixture was then rapidly transferred by a micropipette over the resolving gel and, after placing the comb over it, left to harden. The samples were prepared by getting 100 ? L of the protein sample, 20 ? L of distilled water, and 80 ? L of loading buffer with ? -mercaptoethanol in plastic tubes for the electrophoresis of the samples containing ? -mercaptoethanol. For those samples not containing the latter reagent, 80 ? L of the loading buffer was added. The same procedure was done for 100 ? L of bovine serum albumin. These were then placed in a boiling water bath for 10 minutes after which these were immediately immersed in an ice water bath for 3 minutes. The protein samples used were invertase, albumin, and casein. The loading buffer was prepared by mixing 2. 5 mL of 10% SDS, 2. 5 mL of 0. 625 M Tris-HCl at pH 6. 8, 2. 5 mL of 10% glycerol, and 5. 0 g of 0. 02% bromophenol blue, and diluting to 25 mL with distilled deionized water. Eight tubes were done all-in-all. The gel slabs were then placed in the gel chamber. The gel chambers were then filled with gel running buffer, making sure that the gel was completely immersed. This buffer was prepared by mixing 3. 0 g Tris base, 14. 4 g glycine and 1. 0 g SDS, and diluting to 1 L with distilled deionized water.. The set-up was then placed on a level surface. At this point, the comb was removed in one fluid motion to ensure that the wells would have straight edges. Ten microliters (10 ? L) of the samples with ? -mercaptoethanol was loaded into the wells using a micropipette. With the voltage set at 100 V and the protective electrode covering placed over the set-up, the gel was run until the dye reaches a level of 1 cm above the bottom of the gel slab. This was done again for those samples without the ? -mercaptoethanol. After the gels have been run, the gel slabs were transferred from the glass plates to a flat-bottom container where a small amount of staining solution was added until the slabs were completely immersed. This solution was prepared by mixing 50 mL of methanol, 10 mL of glacial acetic acid, and 0. 25 mg of Coomassie Brilliant Blue R250, and diluting to 100 mL with distilled deionized water. After that, the background staining was removed by several washings of destaining solution. This solution was prepared by mixing 25 mL of 95% ethanol and 5 mL of glacial acetic acid, and diluting to 100 mL with distilled deionized water. Results and Discussion Polyacrylamide Gel Electrophoresis (PAGE) served as an effective tool in the characterization of protein standards and extracts because of the gel’s high resolving power for molecules up to 106 Da, accommodation of larger sized samples, an inert enough matrix with respect to the migrating entities, and physical stability of the matrix (Boyer, 1993). Polyacrylamide gels were prepared by the catalyzed and cross-linked polymerization of the acrylamide-bisacrylamide mixture. The polymerization reaction was facilitated by ammonium persulfate (APS), the polymerizing agent, due to its inherent instability and, hence, its tendency to decay and to give rise to molecules initiating these polymerization. Tetramethylethylenediamine (TEMED) was introduced to catalyze the decay of APS. Figure 1 presents the general equation for the polymerization reaction of the acrylamide-bisacrylamide mixture (Encor Biotechnology, Inc. , 2011). Figure 1. The polymerization reaction of the Acrylamide-bisacrylamide in the presence of ammonium persulfate and TEMED as the polymerizing agent and the catalyst respectively (Thermo Scientific, Inc. , 2011) Polymerization proceeded with the opening of an acrylamide double bond, allowing it to react with another acrylamide to produce a linear polyacrylamide. Cross links were generated through the incorporation of bisacrylamide into the linear polyacrylamides. Since molecular oxygen would react with the free radical sulfate ions (SO42-) thereby inhibiting polymerization, degassing was necessary. Furthermore, the tendency of molecular oxygen to react with SO42- would also be the reason why it would be necessary for PAGE gels to be poured into tubes or between glass plates instead of horizontal apparatuses. However, the degassing step was not done due to the unavailability of a degassing chamber. Isobutanol was added on top of the gel to also prevent the entry and accumulation of O2 (Encor Biotechnology, Inc. , 2011). Gel pore size is inversely proportional to the concentration of acrylamide. Therefore, to generate a broad and efficient range of protein separation, a discontinuous gel system was formulated, having a low acrylamide content on top and a high acrylamide content at the bottom. The capability of Tris-HCl to facilitate the propagation of electric current through the matrix qualified it as an appropriate loading buffer. It allowed the proteins to be drawn by the current through the sieving matrix slab (Thermo Scientific, Inc. , 2011). The polyacrylamide gel electrophoreses set-up had three important features. First, a stacking gel was cast over a resolving gel. Second, the two gel layers had different ionic strengths and pH. Third, the stacking gel had a lower acrylamide concentration and a lower pH. These conditions allowed the protein molecules to first concentrate into a tight band before entering the resolving solution. In this experiment in particular, the charge of the protein was kept uniform all throughout using sodium dodecyl sulfate (SDS), a powerful detergent that would denature the protein and would leave it evenly negatively charged. Also, ? -mercaptoethanol was added to cleave the disulfide bonds, enforcing completely disrupted secondary, tertiary, and quarternary structures. Prior to the loading of the sample, the discontinuous gel system was immersed in a glycine-Tris buffer prepared at pH 8. 8. At this pH, the two form of glycine – its Zwitterion ion and glycinate – would exist in equilibrium. H3N+CH2COO- – H2NCH2COO- + H+(2) When the voltage was turned on, the entry of buffer ions (glycinate and H+) to the stacking gel (pH 6. 8) shifted the equilibrium to the left, increasing the concentration of glycine’s Zwitterion ion, which would have a zero net charge, and therefore, would be electrophoretically immobile. Since the protein molecules would still be anionic at pH 6. , they would replace the nonmobile glycine molecules in order to keep the current running. As such, the relative mobilities of the ions in the stacking gel would be Tris base > protein sample > glycinate. Furthermore, the thin band observed in the upper gel would actually pertain to the protein molecules sandwiched between the Tris-base and the glycinate ions . The resolving gel, on the other hand, had a pH of 8. 8. When the ionic front reached it, the equilibrium of glycine species shifted to the right. The increase in pH and decrease in pore size retarded the movement of proteins and rendered the glycinate ions greater mobility. The relative rates of movement then became Tris-base > glycinate ions > protein samples. From there, it was the mass of the protein molecules that governed their mobility and thus identified them (Boyer, 1993). For qualitative analysis of results, the Coomassie brilliant blue dye (R-250), being the most popular staining reagent for the electrophoresis of protein samples, was used. Its mechanism of binding to the basic and hydrophobic groups of proteins manifested in the dull, reddish-brown to intense blue color change of the solutions. The staining method was started with the water wash of the gel cast to remove the electrophoresis buffers from the matrix. The matrix was then washed with methanol followed by glacial acetic acid to prevent the diffusion of protein bands form the matrix. The treatment with the dye followed. Lastly, destaining measures were employed to get rid of excess dye from the background gel matrix. This would allow a clear visualization of the bands that had formed (Thermo Scientific, Inc. , 2011). Figures 2 and 3 are photographs of the two gels after incubation and subjection to the dye. Figure 2. Photograph of 1st gel Figure 3. Photograph of 2nd gel In figure 2, multiple bands existed. This could suggest that the samples had other components. These could come in the form of other proteins, contaminants, or other impurities. Nonetheless, any of these possibilities suggest one thing; the sample is not pure although there are occasional times when homogeneous samples result to multiple bands due to degradation during the electrophoresis procedure (Boyer, 1993). Also, the identity of the proteins could have been determined if there were standards or â€Å"markings† to compare these bands with. However, there were none. The only information that could be extracted from the photographs could be that the proteins in the samples were heavy that they were only located on the upper half of the gel. Conclusion The separation of biomolecules according to charge, size, and shape through electrophoresis could give significant information such as the purity, molecular weight, and, hence, the identity of the biomolecule. In this experiment, the multiple bands produced in the gel set-ups suggested that the samples were heterogeneous. Their location in the gel suggested that the proteins were relatively heavy ones. To gain more valuable information rom these data, it is recommended that a set of standard solutions be also run on the gel so that they could be used as references for the identification of the proteins in the samples. Also, the protein’s exact molecular size could be determined by preparing a calibration curve from a set of standard solutions of proteins, with of course, known concentration. The curve should be a plot of the logarithm of the molecular weight of the protein versus its mobility in the gel matrix. From this curve, the molecular weight of the protein in the sample solutions could be extrapolated. References 1. Boyer, Rodney. Modern Experimental Biochemistry. Third Edition. San Francisco, USA: Benjamin/Cummings, 1993. Scribd. Web. 29 November 2011. 2. Encor Biotechnology, Inc. â€Å"SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). † Encor Biotechnology, Inc. Protocols. Encor Biotechnology, Inc. , 2011. Web. 30 November 2011 < http://www. encorbio. com/protocols/SDS-PAGE. htm>. 3. Thermo Scientific, Inc. â€Å"SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). † Thermo Scientific, Inc. Protein Methods Library. Thermo Scientific, Inc. , 2011. Web. 30 November 2011 < http://www. piercenet. com/browse. cfm? fldID=21518847-2D72-475F-A5B9-B236EC5B641E >.

Brl Hardy Driving Forces to Become a Global Company

What are the driving forces behind BRL Hardy to become a global company? â€Å"A Global company is an organization that attempts to standardize and integrate operations worldwide in all functional areas. † In general, there are multiple Globalization forces; some of them are: * Industrial: get access to a bigger market to sell the product. * Financial: by emerging worldwide, it is easier to borrow money * Political forces: the raising globalization goes along with the decrease of the importance of the state. Companies can set up their headquarter in different countries, in function of the legislation in those countries. * Technological forces: the new discoveries and the fast evolving technology eases the communication and makes it easier to collect information about foreign/other goods. * Market: when companies globalize, they also become global customers. * Cost: By becoming global, companies can benefit from economies of scale. The company can also locate production in countries where production costs are lower. In early times, the wine industry was very little. There were little village labels and the grapes grew on tiny vineyards. Those factors made the wine industry very agricultural i. e. the harvest was very vulnerable to weather and diseases. On the other side, the wine business had very few multinational companies and therefore very few true global brands. This made BRL Hardy think about expanding its business to multiple locations over the world and become one of the world’s first global wine companies. The first company on the market has a big chance to become one of the biggest companies in his sector. By breaking the habit of growing and selling only its own wine, Hardy was able to build the scale necessary for creating strong brands and negotiating with retail stores. In 1882, BRLH won his first international gold medal at Bordeaux. Winning a price creates a certain reputation, which makes it more likely that the wine will sell if the company becomes a global company. Also, the company was Australia’s largest winemaker, and one of the most respected. Next to this, Australian wine was becoming a trend, and the demand from new customers in nontraditional markets grew rapidly. All this were driving forces to become a global company. Sources: http://www. slideshare. net/gugaslide/global-business-presentation http://www. slideshare. net/RealRedOne/harvard-business-school-brl-hardy-globalizing-an-australian-wine-company http://www. businessschoolnetherlands. com/files/bsn-article_marius-leibold_business-model-innovation_1. pdf http://www. andidas. com/academic/lse_coursework/MN498%20-%20Tesco%20Internationalisation_by_andidas. pdf http://scholar. sun. ac. za/bitstream/handle/10019. 1/3328/Ewouba-Biteghe,%20BS. pdf? sequence=1 http://en. wikipedia. org/wiki/Globalization