Inhibition Effectiveness of Au Compounds

Inhibition Effectiveness of Au Compounds Function of the â€Å"Guiding Bar† on Inhibition Effectiveness of Au Compounds on Thioredoxin Reductase 3 Qi Liu Introduction Mammalian thioredoxin reductase (TR) is an Nicotinamide adenine dinucleotide phosphate (NADPH) dependent flavoprotein oxidoreductase, which is involved in reducing the disulfide of thioredoxin (Trx) using NADPH (Figure 1). Trx can reduce many important proteins, such as ribonucleotide reductase (RNR), which produces deoxyribonucleotides for DNA synthesis1, 2. Thus, TR and Trx play an important role in maintaining proteins in their reduced state, which involves the regulation of cellular redox reactions, proliferation, and differentiation3. In addition, Trx and TR is overexpressed in a lot of aggressive tumors, and the tumor progression and metastasis appear to be dependent on the Trx system, because tumor cells need constant DNA synthesis. However, tumor progression and metastasis were dramatically reduced when TR knockdown cells were injected into mice4. Thus, the TR was proposed to be a new anticancer drug target5. In mammals, there are three different TRs which have been characterized: the cytosolic form TR1, the mitochondrial form TR3, and thioredoxin glutathione reductase (TGR, also known as TR2)6. All of these TRs are selenoproteins with a selenocysteine (Sec) at their C-terminal active site. In the reaction mechanism of TR, the selenolate of Sec acts as an electron donor to Trx, also selenium can accept electrons from the N-terminal redox center of TR6. This redox center is located on a flexible arm, which is solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development1. A number of recent reports suggest that gold-based drugs have strong inhibition ability on TRs, which could be from the direct coordination between Au and the active site Sec on TRs, so that the selenolate group is blocked7. Interestingly, the gold compounds,Bis[1,2-bis(dipyridylphosphino)ethane]gold(I) chloride ([Au(d2pype)2]Cl) and Bis[1,3-bis(di-2-pyridylphosphino)propane]gold(I) chloride ([Au(d2pypp)2]Cl), effect the inhibition effectiveness on human TR1(hTR1) and human TR3 (hTR3) differently (Figure2). The precise molecular mechanism of TR inhibition by gold compounds has not been elucidated. Thus, studies on inhibition of TRs by gold-based drugs are necessary for designing new cancer inhibitors. Available crystal structures of TRs include hTR1, rat TR1 (rTR1), mouse TR3(mTR3), DmTR (thioredoxin reductase from D. melanogaster), and PfTR (thioredoxin reductase from P. falciparum) enzymes9. A recent study showed the crystal structure of hTR1 with its substrate thioredoxin, in which the C-terminal tail of hTR1 was stabilized by a â€Å"guiding bar†, so that the flexible C-terminal tail can be observed (Figure 3). The guiding bar was first proposed in one of the structure studies of hTR1, and is composed of three amino acids: Trp407, Asn418, and Asn419. It functions to suppress random motions and positions the C-terminal tail in catalytically competent position through hydrogen bonding interactions9. However, the crystal structure of mTR3 does not show the C-terminus because of the random motion of the C-terminal without control from the guiding bar. Furthermore, the sequence comparison between hTR1 and hTR3 shows the absence of the guiding bar in TR3 (Figure 4). Research question Though inhibition of gold compounds on TRs has been studied, the function of the guiding bar on the gold compounds inhibition effectiveness on TRs has never been brought up to the studies of inhibition effectiveness on TRs. The goal of this proposal is to investigate the role of the guiding bar on the inhibition ability of gold based compounds on hTR1 and hTR3. The guiding bar will be constructed on hTR3 by mutations of K432W, A443N and S444N. Biophysical characterization will be applied to observe the presence of the C-terminus on TR3 after mutagenesis. My hypothesis is the C-terminal tail of the crystal structure of mutated TR3 will be observed, because the random motion of the C-terminus is reduced by restriction from the guiding bar. Then, two gold compounds, [Au(d2pype)2]Cl and [Au(d2pypp)2]Cl , will be applied to test the inhibition difference on normal TR3 and mutated TR3. I expect to see that the inhibition on mutated TR3 could be stronger than that on normal TR3. Significance of Proposed Research The thioredoxin system plays an important role in the intracellular redox enviornment and is composed of Trx and TR10. Proposed studies will give an insight into the function of the guiding bar on different TR enzymes. The guiding bar is expected to suppress the random motion of the C-terminal tail so that the redox center Sec on the C-terminus can have better coordination with the gold compounds. The coordination between metal and Sec will tightly block the Sec redox center, so the intracellular redox balance will be disturbed and result in potent TR inhibition4. Thus, the presence of the guiding bar can help TR to be a better drug target. Proposed studies Enzyme Expression and Purification Recombinant human TR1 and TR3 will be cloned into E. coli BL21(DE3) cells, and the growth and purification will be done as described previously.11 Mutation of Human TR3 In order to study the impact of the guiding bar residues on TR catalysis, the proposed mutations will be constructed on hTR3 with K432W, A443N, and S444N. These three mutation sites are chosen based on the sequence alignment of hTR1 in the guiding bar region of Pro376-Tyr422 (Figure 3), in which the critical amino acids, Trp407, Asn418 and Asn419, have the function of limiting random motion of the C-terminus. Thus, the same function of the guiding bar on hTR3 is expected to be observed via specific site mutation. Mutations will be introduced by the Quick Change mutagenesis method and confirmed by DNA sequencing. Structural Studies of Mutated Human TR3 The C-terminal tail in human and rat TR1 could be observed by X-ray crystallography, because the guiding bar limits its random motion. However, because the guiding bar is not present in mouse TR3, the C-terminal tail is too mobile to generate sufficient electron density without restriction from the guiding bar. This leads to the absence of the C-terminal tail in the crystal structure of mouse TR39. The mutated hTR3 after insertion of the guiding bar will be studied by X-ray crystallography. The presence of an ordered C-terminal tail in the crystal structure and the interaction between the guiding bar and the C-terminal tail will suggest restricted motion of the C-terminal tail. I expect to see that the interaction of the guiding bar on the C-terminal tail will improve the inhibition effectiveness of Au compounds on hTR3. Electrospray ionization mass spectroscopy (ESIMS) Characterization ESI is an ionization technique which is used to detect high molecular weight molecules, such as proteins, peptides and other macromolecules, so ESI MS is an important technique for studying a complex biological sample9. Instead of fragmenting the macromolecules into smaller charged particles, this process turns the macromolecules into small droplets by ionization, and these droplets are further desolvated into even smaller droplets, which creates molecules with attached protons12. As a putative target for anticancer metallodrugs, the selenoenzyme TR is the drug target for gold compounds, such as [Au(d2pype)2]Cl and [Au(d2pypp)2]Cl. The TR inhibition is thought to occur through direct binding of the gold to the active site Sec following ligand substitution. So, TR3 and mutated TR3 will be separately incubated with the two gold compounds mentioned above for 30 mins at room temperature, and then the products will be analyzed by EIS-MS. The results will be compared and are expected to explain the binding of the gold onto the hTRs. I hope to see the gold compounds have better binding on mutated hTR3 than normal hTR3. This can be explained as the guiding bar can reduce the motion of flexible C-terminal tail, so that the gold compounds can bind onto the Sec of mutated hTR3 easier than the normal one and cause more inhibition on the mutated hTR313. TR Inhibition Assay The solvent-accessible selenolate group, arising from enzyme reduction, very likely constitutes a high affinity binding site for gold compounds. Tightly blocking the active site Sec through metal coordination should result in potent TR inhibition9. Thus, after ESI-MS characterization, the TR inhibition study will be performed with the two different gold compounds, [Au(d2pype)2]Cl and [Au(d2pypp)2]Cl, on hTR1, hTR3 and mutated hTR3 respectively. In addition to Trx, some low molecular weight disulfide-containing substrates, including 5,5†²-dithiobis-(2-nitrobenzoic acid) (DTNB), lipoic acid, and lipoamidem, can also be reduced by TRs9. Here, DTNB would be chosen as the substrate for TR inhibition assay to keep consistent with the previous studies from other groups9. The NADPH dependent TR catalyzed reduction of DTNB will be monitored and determined by the increase in absorbance at 412nm. Inhibition of hTR1, hTR3 and mutated hTR3 will be done by using different concentrations of go ld compounds ([Au(d2pype)2]Cl and [Au(d2pypp)2]Cl). The inhibition results from different TRs and gold compounds will be compared. Based on the assumption that the guiding bar can suppress the motion of the C-terminal tail, greater inhibition effectiveness on mutated hTR3 than that on normal hTR3 is expected to be seen. This will show that the C-terminus is limited by the guiding bar on mutated hTR3 helps the gold compounds inhibit the active site Sec more. Binding Studies by Isothermal Titration Calorimetry (ITC) ITC is a technique used to determine the thermodynamic parameters of interaction in solution. It can be applied to study the interactions between protein-protein, enzyme-inhibitor, protein-small molecules, protein-DNA, and so on. Thus, the binding between gold compounds and the three TRs (hTR1, hTR3 and mutated hTR3) can be investigated by ITC. Applying this method will tell us how well the inhibitors, the gold compounds, bind to TRs. The binding affinity (Ka) could show how strongly the gold compounds and TRs will bind, and the higher Ka, the stronger the binding. In addition, enthalpy changes (ΔH) can tell the amount of energy released or obtained. Then, the entropy change (ΔS) and Gibbs energy changes (ΔG) can be determined by the following equation (1): ΔG = -RTlnKa = ΔH-TΔS (1) Comparison of the binding parameters between gold compounds ([Au(d2pype)2]Cl and [Au(d2pypp)2]Cl) and TRs (hTR1, hTR3 and the mutated hTR3) will give more information about the function of the guiding bar on the inhibition effectiveness of TRs. The higher Ka of hTR1 compared to normal hTR3 is expected to be seen, because the absence of guiding bar in hTR3 will lead to a flexible C-terminal tail, which will give a lower inhibition. In addition, Ka of the mutated hTR3 is expected to show higher binding affinity than the normal hTR3, because the guiding bar mutated on hTR3 will help reduce the random motion of the C-terminal tail, so that the redox center Sec can be easily inhibited by the gold compounds. Thus, the guiding bar can help improve the inhibition effectiveness of gold compounds on mutated hTR3. Conclusion Through mutation and structural studies, the presence of the C-terminal tail which is restricted by the inserted guiding bar is expected to be seen in the mutated hTR3. The ESI-MS characterization and binding study can give information about if the gold can successfully bind to the specific active site on the mutated hTR3. Finally, a better understanding of the inhibition effectiveness will be tested and compared by the inhibition assay with hTR1, hTR3 and mutated hTR3. Thus, a better understanding of the function of the guiding bar in the TR system will give an insight into the effect of the guiding bar on the drug inhibition effect on TR3 so that it can become a better drug target. References O. Rackham., A. M. Shearwood., R. Thyer., E. McNamara., S. M. Davies., B. A. Callus., A. Miranda-Vizuete., S. J. Berners-Price., Q. Cheng., E. S. J. Arner and A. Filipovska,Free Radical Biol. Med. 2011, 50, 689–699 Lothrop A. P.,Snider G. W.,RugglesE. L., PatelA. S., LeesW. J., and HondalR. J., Biochemistry.2014,53, 654–663 Fritz-Wolf K, Kehr S, Stumpf M, Rahlfs S, and Becker K., Nat Commun.2001, 2, 383 Fritz-Wolf K., Urig S., Becker K., J. Mol. Biol. 2007, 37, 116–127 Lothrop, A. P., Snider, G. W., and Hondal, R. J., Biochemistry. 2014, DOI: 10.1021/bi400651x Pratesi A., Gabbiani C., Michelucci E., Ginanneschi M., Papini A. M., Rubbiani R., Ott I., Messori L., J. Inorg. Biochem. 2014 Gabbiani C., Mastrobuoni G., Sorrentino F., Dani B., Rigobello M. P., Bindoli A., Cinellu M. A., Pieraccini G., Messori L. and Casini A.,Med. Chem. Commun. 2011,2, 50–54 Lu J.,Chew E. H.,Holmgren A.,Proc. Natl. Acad. Sci.2007, 104,12288 Yoo M.H., Xu X.M., Carlson B.A., Gladyshev V.N., Hatfield D.L., J BiolChem, 2006, 281, 13005–13008. Arnà ©r E.S.J., Holmgren A., Eur. J. Biochem, 2000, 267, 6102–6109 Gromer, S., Urig, S., and Becker, K., Med. Res. Rev. 2004, 24, 40-89 http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Mass_Spectrometry/Mass_Spectrometers_(Instrumentation)/Electrospray_Ionization_Mass_Spectrometry Lu, J., Chew, E.H., Holmgren, A., Proc. Natl. Acad. Sci.,2007, 104, 12288–12293. Berners-Price S. J., inBioinorganic Medicinal Chemistry, ed. E. Alessio, Wiley-VCH, Weinheim Berners-Price S. J. and Filipovska A.,Metallomics, 2011,3, 863–873

The Minimum Wage For Restaurant Workers Must Be Raised Essay -- Argume

"It is but equity...that they who feed, clothe and lodge the whole body of the people, should have such a share of the produce of their own labor as to be themselves tolerably well fed, clothed and lodged." -- Adam Smith, The Wealth of Nations, 1776 The restaurant server is one of the most popular jobs in the country and according to the National Restaurant Association, currently, 1 in 12 Americans work in the restaurant industry and about 50 percent of all adults have worked in the industry at some point during their lives. The industry is clearly very large with over 13 billion workers, but overall, workers in the industry do not get paid very well. According to the U.S. Department of Labor (DOL), the average annual salary for a server is just over $20,000. In fact, servers are twice as likely to use food stamps as the rest of the U.S. workforce and three times as likely to be living in poverty. The main reason for this is the existence of the tipped minimum wage, which since 1991 has stayed set at $2.13 per hour. Not many people even realize that the tipped minimum wage exists, but according to the DOL, servers in 43 states get paid less than the regular minimum wage hourly based on the assumption that the rest of their wages will come from customer tips. In fact, 22 states pay their tipped workers less than $3 per hour. Although tips can often lead to servers making well over regular minimum wage per hour, overall, tips are very inconsistent and are completely dependent on restaurant customers. Not only does the customer decide how much to tip based on his/her enjoyment of the dining experience, but also servers need busy restaurants in order to make good money. There is an ebb and flow to the restaurant busin... ...y-income-fine-dining-servers-20195.html>. Myotte, Maria. â€Å"Op-Ed: Restaurant Servers Can’t Live on $2.13 an Hour.† Yahoo! News. Yahoo!, 11 Dec. 2013. Web. 30 Oct. 2014. . National Restaurant Association. 2013 Restaurant Industry Pocket Factbook. Rep. National Restaurant Association, 2013. Web. 30 Oct. 2014. . Nayak, Rajesh, and Paul Sonn. Restoring the Minimum Wage for America’s Tipped Workers. Rep. National Employment Law Project, 2009. Web. 30 Oct. 2014. . U.S. Department of Labor. Minimum Wages for Tipped Employees. Rep. U.S. Department of Labor Wage and Hour Division, Dec. 2011. Web. 30 Oct. 2014. .

Black People and Roberta Essay

In Toni Morrison’s â€Å"Recitatif,† the story is about two girls, Twyla and Roberta. They grow up in an orphanage because their mothers could not care for them. Morrison makes it clear the girls come from different ethnic backgrounds but never states which one is black or white. At one point in the story Twyla comments, â€Å"We looked like salt and pepper. † I grew frustrated with the story and had to read it several times. I could never determine who was black and white and the lesson I learned should have been it doesn’t really matter. The story begins with Twyla’s mother dropping her off at the orphanage. She meets Roberta and they become best friends. The bond they share occurs because they were not considered real orphans. They were abandoned kids unlike the other children whose parents had died. One of the last times the girls see each other was the day of a visitation. On that night, Twyla’s mother was wearing â€Å"those tight green slacks that made her butt stick out. † Many people have labeled blacks as having larger butts. She could have been black, she could have been a heavy white woman with a large butt, or a Hispanic woman like me. But I automatically stereotyped and went with Twyla has to be black. During the visitation Roberta’s mother â€Å"had brought chicken legs. † Twyla notices Roberta does not eat the chicken legs. I always thought black people liked chicken more than white people which means Roberta was white since she did not eat the chicken. Or maybe she just wasn’t hungry. Shortly after that visitation Roberta’s mother came to take her home, leaving the girls devastated. They see each other several times throughout the years. At their first meeting, Roberta was rude and distant because she was high. Roberta tells Twyla she is on the way to see Jimi Hendrix. Hendrix was an infamous black guitarist. I thought at this point Roberta has to be black. However Hendrix’s band was interracial with a diverse audience. Roberta could have been white due to the diverse audience. I am a huge Hendrix fan and I am not black so why would I think Roberta is. Twelve years later they meet again at a grocery store. Roberta married a rich man and was extremely friendly to Twyla. Twyla cannot hold back her emotions and asks Roberta about the last time they saw each other. Roberta shrugs it off, â€Å"Oh, Twyla, you know how it was in those days: black—white. You know how everything was. † I can relate to this. In 1980, the Cuban Mariel Boat Lifts came over bring thousands of Cubans. I am Cuban but I was born here. Kids I had known since kindergarten treated me as if I just come over on the boats. It had a lasting effect on me and matured me beyond my years. The third time they meet is at the school where their children attend. Roberta and other mothers were picketing because they did not want their kids to be segregated. This led to a fight severing any last chance of a friendship for them as it would not be resolved until Twyla and Roberta meet for a final time. As the story ends I do not get a sense of closure. The question of which girl is white or black remains unanswered. It opened my eyes and made me question how prejudice I really am. I try to not stereotype as a result of what I went through as a child but I found myself doing just that. I can understand why Morrison wrote the way she. I am not sure what her goal was overall but to me it seemed as if she were teaching me about prejudices. â€Å"Recitatif† challenged me to not judge either girl by their race but accept them for who they are. In the end, what difference did it really make about the girls’ races? The story is about how their friendship develops and then deteriorates. Nothing more; nothing less.

The Epic of Gilgamesh Explication Assignment - Free Essay Example

Sample details Pages: 7 Words: 1989 Downloads: 8 Date added: 2019/07/29 Category History Essay Level High school Tags: Gilgamesh Essay Did you like this example? Then he called to the man Gilgamesh, he called to the child of the gods: Why have you come so great a journey; for what have you travelled so far, crossing the dangerous waters; tell me the reason for your coming? Gilgamesh answered, For Enkidu; I loved him dearly, together we endured all kinds of hardships; on his account I have come, for the common lot of man has taken him. I have wept for him day and night, I would not give up his body for burial, I thought my friend would come back because of my weeping. Since he went, my life is nothing; that is why I have travelled here in search of Utnapishtim my father; for men say he has entered the assembly of gods, and has found everlasting life. Don’t waste time! Our writers will create an original "The Epic of Gilgamesh Explication Assignment" essay for you Create order I have a desire to question him concerning the living and the dead. The Man-Scorpion opened his mouth and said, speaking to Gilgamesh, No man born of woman has done what you have asked, no mortal man has gone into the mountains; the length of it is twelve leagues of darkness; in it there is no light, but the heart is oppressed with darkness. From the rising of the sun to the setting of the sun there is no light. Gilgamesh said, Although I should go in sorrow and in pain, with sighing and with weeping, still I must go. Open the gate of the mountain. And the Man-Scorpion said, Go Gilgamesh, I permit you to pass through the mountain of Mashu and go through the high ranges; may your feet carry you safely home. The gate of the mountain is open. The Norton Anthology, World Masterpieces, Gilgamesh, 32. In this passage, Gilgamesh, after the death of Enkidu, wants to go to Utnapishtim who has achieved immortality and wants to gain immortality for himself. Gilgamesh has to go through the great mountains of Mashu and the Man-Scorpion was guarding it. The Man-Scorpion according to the text is half man and half dragon. Gilgamesh, 32. This shows the guard of the mountains as a terrifying creature that people cannot go through because of fear. The Man-Scorpion resisted from letting Gilgamesh go at first but eventually let Gilgamesh pass because of his unbreakable determination of meeting Utnapishtim to achieve immortality. When the Man-Scorpion calls Gilgamesh, he uses the terms man and child. The use of the term man shows that the Man-Scorpion was not afraid of Gilgamesh and had a higher position and power than him even though Gilgamesh is two third god and has defeated multiple enemies before meeting the Man-Scorpion. The word man also in a way foreshadows Gilgameshs fate, that even though he was searching for immortality, he would not find it because men are mere mortals and have to die. Another term that the Man-Scorpion uses is child. The use of the word child portrays the childlike nature of Gilgamesh. Even though Gilgamesh went through changes since being with Enkidu, he is still called child because he has little knowledge of things such as death just like a child. The only time Gilgamesh experienced death personally was when his friend, and brother in arms, Enkidu, died. This affected Gilgamesh, as he did not experience death closely before. Another thing that can be seen from this passage is Gilgameshs selfishness. Gilgamesh was asked why he had travelled so far, he said that it was out of his love for Enkidu. He wanted to ask Utnapishtim about the living and the dead and wanted to know how to achieve immortality. Enkidus death made him realize that his life could potentially come to an end as well no matter how strong the gods made him. However, that passage also shows that Gilgamesh did truly love Enkidu. Before Enkidu died, they did a lot together and according to the passage, Gilgamesh told this to the Man-Scorpion, I loved him dearly, together we endured all kinds of hardships. The word dearly means, with affection: fondly and also heartily, earnestly. dearly. Merriam-Webster.com. 2018. https://www.merriam-webster.com (30 November 2018). This shows the amount of love Gilgamesh had for Enkidu and relating it back to one of Gilgameshs dreams where he saw a meteor and an axe, and he said to his mother that he loved them like a woman. Gilgamesh, 16 A man loves a woman more affectionately than he would love his brother and so Gilgamesh saying that he loved Enkidu dearly shows that they had a very strong relationship that was beyond just being brothers in arms. The part of the sentence when Gilgamesh says, we have endured all kinds of hardships can be seen as Gilgamesh talking about the difficult battles that him and Enkidu have been through but another not so obvious meaning behind it is that it can be seen as a husband and wife going through, or enduring hardships in their life. Even though that meaning is not obvious to readers at first, through the relationship that Gilgamesh and Enkidu had before Enkidu died, shows how much Gilgamesh loved Enkidu and was willing to make a long and difficult journey for him. This reason, however, contradicts Gilgameshs selfish reason to go to Utnapishtim and gain immortality so that he himself does not die. Enkidus body had been taken away for burial by people whom Gilgamesh describes as, the common lot of man. The meaning of the word common in this context is to be, characterized by a lack of privilege or special status. common. Merriam-Webster.com. 2018. https://www.merriam-webster.com (1 December 2018). The use of the word common to describe the people shows that Gilgamesh still thought of himself highly, and above others, so the people that took away Enkidus body were just common people without any special privilege or status unlike Gilgamesh who was a two-thirds god. Gilgamesh did not want Enkidus body to be taken away and did not want Enkidu to die, so he was crying day and night and he thought his crying could bring back Enkidu. This again shows Gilgameshs childlike knowledge of death. Once someone is dead, he or she cannot return back to life, but Gilgamesh did not know that and so he was weeping for Enkidu to try to bring him back. This can be related to the beginning of the passage when Gilgamesh was called a child by the Man-Scorpion, which shows that even though Gilgamesh was higher than others in strength and status, he had knowledge of things such as death like a child. However, this little knowledge of death also shows that Gilgamesh previously did not care about it much , neither others death or his own, but when Enkidu, someone extremely dear to him died, it was as if he was shaken up, and came to the realization that someday it could be his own fate. After the death of Enkidu, Gilgamesh felt as if he lost purpose in life, which is why he says to the Man-Scorpion, Since he went, my life is nothing. The pronoun he is referring to Enkidu and the use of the word nothing shows that Gilgamesh felt empty as if the loss of Enkidu made him lose something of his own since they were so close to each other. He did not know what to do in his life at that point and that is the reason he gives to the Man-Scorpion to let him pass through the mountains to meet Utnapishtim. Gilgamesh refers to Utnapishtim as his father, as he says to the Man-Scorpion, that is why I have travelled here in search of Utnapishtim my father. It is not clear how Gilgamesh is referring to Utnapishtim as a father in this context. It could be as his actual father or father in meaning someone higher than him, such as addressing gods or kings. It could be that Gilgamesh is referring to Utnapishtim as a father because he had entered the assembly of gods by gaining immortality . Utnapishtim gained immortality in when Uruk was flooded by the god Enlil, but as Utnapishtim survived he was blessed with immortality by Enlil. Gilgamesh, 37-39. Gilgamesh desired for this everlasting life as well. He says to the Man-Scorpion, I have a desire to question him concerning the living and the dead. Gilgamesh wants to ask questions to Utnapishtim and find out the secret of life and death and how he could be immortal. The use of the phrase I have a desire, rather than I want to, shows how much Gilgamesh cares about this subject, life, and he emphasizes his will to ask and know about the living and dead by using the word desire. To this, the Man-Scorpion said that no man born of woman had ever done what Gilgamesh had asked to do. He also said that no mortal man had ever gone through the mountains. By saying no man born of a woman and the word mortal man, the Man-Scorpion asserts his position over Gilgamesh, that he is more powerful than him, but it also foreshadows Gilgameshs fate that he will die as a mortal. The use of the term mortal also strengthens the Man-Scorpions argument that Gilgamesh is mortal and since no mortal man had g one through the mountains, Gilgamesh could not either. The Man-Scorpion tries to input more fear into Gilgamesh by describing the path inside the mountain. The path had no light, there was twelve leagues of darkness, and he also says, the heart is oppressed with darkness. The entire length of the journey would take Gilgamesh days because the Man-Scorpion says that from when the sun rises to when it sets, there is no light in the mountain. The path seems to be a very long and difficult path but what is most interesting is when the Man-Scorpion says the heart will be oppressed with darkness. This could mean that fear would not let someone complete the journey through the mountain as it is so dark. The word oppress means to crush or burden by abuse of power or authority and also to burden spiritually or mentally. oppress. Merriam-Webster.com. 2018. https://www.merriam-webster.com (8 December 2018). This shows that Gilgamesh would be mentally oppressed in the darkness, as he will not be able to see or comprehend his surroundings. Gilgamesh is not moved by the Man-Scorpion, he is still determined to go through the mountain to meet Utnapishtim. He says that he would go in sorrow and in pain, sighing and crying, but he still had to go no matter what. This bold determination most likely comes from him not wanting to die, and his sorrow, pain, and weeping come from the death of his dear Enkidu. The word sorrow in this context means, deep distress, sadness, or regret especially for the loss of someone or something loved. sorrow. Merriam-Webster.com. 2018. https://www.merriam-webster.com (14 December 2018). This definition of sorrow confirms that one of the pushes for Gilgamesh to go to Utnapishtim was the death of Enkidu. Yet there is a conflict with another reason for Gilgamesh to go to Utnapishtim, which is his selfish will to become immortal. This conflict can, however, be resolved when Gilgamesh later goes to Utnapishtim and he gets knowledge of the plant which restores youth. When Gilgamesh gets this plant, he not only wants to use it for himself but also give it to other old men to restore their lost youth. Gilgamesh, 40-41. This shows a change in Gilgamesh from being very self-centered in the beginning to caring about others as well. As the Man-Scorpion, the guard of the mountains permits Gilgamesh to go through he says, may your feet carry you safely home, indicating that at the end of the journey, Gilgamesh would be at a place which would be his home. Somewhere he can rest at ease. This foreshadows Gilgameshs death as it could mean that his home is his afterlife, where he can live eternally at peace.