Racial Profliling Essay Essays - Offender Profiling,

Racial profiling must be stopped this is something that people around the world have had to deal with more and more. Stop stereotyping people do not make and assumption based on the color of someone's skin their ethnicity or religion everyone must be treated just as equal as other men and other women. In the united states this is a very big problem mainly because people of color are targeted by the law enforcement. All over the U.S this is a recurring problem and their has been stories of people of color not even being able to walk on the street or not being able to drive the vehicle that they bought without law enforcement stopping them or checking who the car is registered too. This is something that shouldn't even be a problem this outright just should not happen. He who gives himself entirely to his fellow-men appears to them useless and selfish; but he who gives himself partially to them is pronounced a benefactor and philanthropist (Thoreau C.D 14). This speaks to me because it is saying that a if you give someone all your information than you are useless after and selfish if you do not but if you give small points of yourself than they become more intrigued and will see you as useful to them . This is the same case with people like police officers they ask who you are and what you are doing and they want that information to see if you really are a bad person or not but the truth is that in the first place they probably shouldn't have stopped you at all but because of your appearance they make an assumption about you. Think about it is the world really equal because no one is treated fairly especially with law enforcement. When was the last time you heard a person of color be the same as the common man it's not right and it needs to change. All through history there has been racial profiling history has showed us this with the Fugitive Slave Act what is this you might ask well this made it so that if you were a slave that had escaped and left to a free state you must return to your owner. This caused many people of color to be taken if if free they would have been abducted taken from their family and get enslaved. How can we allow such things to happen. How can the United States be proud of this when we have not even been able to bring equality for all . Think Martin Luther King, great man fought for civil rights and was very peaceful but was treated very differently by law enforcement and by government because he was a colored man we speak of him to this day because of the change he has brought to our world it was beautiful it warmed hearts and gave hope even to people that we not of color they joined together and did something good but it is like we have came back to square one in a way. Law Enforcement is not the only thing that racial profiles you also see this in school if you believe it or not it is up to you. Teachers also make assumptions on the people they see the staff does the same but you may not notice it. There was a study and it showed that black student were about 4 times more likely more to get suspended or expelled can you tell me this is not partially due to racial profiling and everyone hates to see it especially the parents of these children. Racial profiling of colored students is just terrible we must change this because think about the people who aren't doing wrong but are put into the same category of people that are actually bad just because of the color of their skin. We must make a change and it all starts with us becoming better people.Law enforcement must change how they see people and should start to see people as one and not give people of color unequal treatment. In Conclusion to stop racial profiling not only do you need to change the

Sample Note Writing For Argumentative Essay With Evidence With Annotating Information

Sample Note Writing For Argumentative Essay With Evidence With Annotating InformationAn effective argumentative essay is one that convinces the reader of the significance of a student's ideas, and why the argument should stand the test of time. This is usually done with a well-developed argument with facts or other supporting data, supported by citations and supporting reasons. While there are many resources to help students in writing their thesis statements, there are still few resources that can offer guidelines for using an annotated thesis statement for argumentative essay with evidence to annotate information. These can be found at many sites for college students online.The first thing to remember is that there is no wrong way to annotate your own document. An annotated thesis statement for argumentative essay with evidence to annotate information is always best used as a supplement to your paper. You will get more information out of the annotated thesis statement for argumenta tive essay with evidence to annotate information when you add your own content to it. This will make your argument more compelling than if you were just trying to keep the paper short.To give you an example, you might start by putting in a piece of your own personal opinion. Or you might have facts or other supporting data that will back up your own argument. Having your own document is not a bad idea; after all, you have more control over what is being written and who is making the final decisions on what goes into the final product.If you are going to put your own content in your thesis statement for argumentative essay with evidence to annotate information, you have to make sure that your annotation is written in such a way that it can easily be read. The best way to do this is to use a hyphenated heading. The important thing is to make sure that your annotated thesis statement for argumentative essay with evidence with annotating information has enough space for the best type of annotation possible.Another way to make sure that your thesis statement for argumentative essay with evidence with annotating information has enough space is to use headers for paragraph headers. Header boxes will give you ample space to annotate your own content. Headers will also give you the opportunity to easily add other supporting data such as a bibliography or a list of citations. Many thesis statement for argumentative essay with evidence with annotating information sites will also provide headers for citation boxes, which you will be able to use to include your citations.Finally, when writing your thesis statement for argumentative essay with evidence to annotate information, it is good to check the content of the original paper and check whether or not the annotation is similar to the content of the paper. This will give you a good idea of whether or not you need to write your own content in the thesis statement for argumentative essay with evidence to annotate informatio n.An annotated thesis statement for argumentative essay with evidence with annotating information can make a good supplemental source to your original paper. You should always be sure to use a title or subhead to provide space for your own content. Using a title will also let you easily associate your own content with the original content of the paper.

Free Essays on An Occurance At Owl Creek Bridge

Throughout the story, we are able to see of different of a world Ambrose Bierce lived in compared to the one that we know. This is the first part of the story that noticed, because in today’s world, no man would ever be hung, let alone executed for tampering with a bridge. Peyton Farquhar grew up as a rich southerner. He had everything that a man could want at that point in time: a wife, children, land and slaves. However, he had always felt something was missing. Due to the fact he was unable to fight in the army, he did not feel like he was really a man. This is why he was willing to anything as â€Å"no service was too humble to him to perform in aid of the south, no adventure too perilous for him to undertake if consistent with the character of a civilian who was a soldier at heart†. Farquhar was blinded by his enthusiasm at it ultimately resulted in his demise. His actions to tamper with the bridge did not completely end in failure. The illusion of escape and heroism that he went through prior to his death, was the most alive he has ever felt. Looking back at Farquhar imagined journey back to his house, you begin to realized the pains and emotions that we was experiencing were more of a man hanging, than of one that just escaped from one. The first example is when Farquhar enters the forest and is surprised to see how dense the forest was, as â€Å"he had not known that he lived in so wild a region† and the stars were in an unfamiliar pattern that night as well. He also refers to the pain he experiences in his neck and his were feeling â€Å"congested† and he could not close them. This is because there is great force being applied to his neck and head from the noose. Farquhar was also because to experience thirst so great that his tongue was beginning to swell, but his tongue was really swelling from the pressure applied by the rope. He began to walk on the untraveled avenue, which symbolizes the avenue of death, and could no lo... Free Essays on An Occurance At Owl Creek Bridge Free Essays on An Occurance At Owl Creek Bridge Throughout the story, we are able to see of different of a world Ambrose Bierce lived in compared to the one that we know. This is the first part of the story that noticed, because in today’s world, no man would ever be hung, let alone executed for tampering with a bridge. Peyton Farquhar grew up as a rich southerner. He had everything that a man could want at that point in time: a wife, children, land and slaves. However, he had always felt something was missing. Due to the fact he was unable to fight in the army, he did not feel like he was really a man. This is why he was willing to anything as â€Å"no service was too humble to him to perform in aid of the south, no adventure too perilous for him to undertake if consistent with the character of a civilian who was a soldier at heart†. Farquhar was blinded by his enthusiasm at it ultimately resulted in his demise. His actions to tamper with the bridge did not completely end in failure. The illusion of escape and heroism that he went through prior to his death, was the most alive he has ever felt. Looking back at Farquhar imagined journey back to his house, you begin to realized the pains and emotions that we was experiencing were more of a man hanging, than of one that just escaped from one. The first example is when Farquhar enters the forest and is surprised to see how dense the forest was, as â€Å"he had not known that he lived in so wild a region† and the stars were in an unfamiliar pattern that night as well. He also refers to the pain he experiences in his neck and his were feeling â€Å"congested† and he could not close them. This is because there is great force being applied to his neck and head from the noose. Farquhar was also because to experience thirst so great that his tongue was beginning to swell, but his tongue was really swelling from the pressure applied by the rope. He began to walk on the untraveled avenue, which symbolizes the avenue of death, and could no lo...

What Makes a Good Villain Heres Your 15-Item Checklist

What Makes a Good Villain Heres Your 15-Item Checklist What Makes a Great Villain? Your Checklist for Writing a Good Bad Guy Nothing makes your hero more heroic than a worthy opponent. So don’t shortchange your villain. Spend every bit as much time crafting him as you do your lead character, if you want your story to work. (Though I will use male pronouns throughout, this applies equally if your main character is a heroine or your villain is female.) Too many novelists give plenty of care to every other element of their story, then create what they consider a deliciously evil villain and wonder why the package seems to fall flat. Often it’s because the bad guy is only that: bad. He’s from Central Casting and might as well be starring in a melodrama, complete with black top hat, cape, and handlebar moustache so we readers can boo and hiss his every entrance. Every other character is real and nuanced and believable, but the second-most important lead spoils the reader’s whole experience. Need help writing your novel?Click here to download my ultimate 12-step guide. Motivation: The Secret Sauce for Creating a Great Villain Don’t let the word scare you. Motivation doesn’t have to be some nebulous theatrical concept tossed about by method actors trying to get into character. It simply means your bad guy needs a reason for being the person he has become. If he isn’t working, it’s because you’ve made him the villain only because he’s a bad person. He does evil things because he’s evil. That’s too easy. Change your thinking. Try something revolutionary. If you just can’t understand truly villainous people, try this: Put yourself in their place. â€Å"Wait!† you say. â€Å"I’d rather see myself as the hero, doing the right thing because it’s the right thing, rising to the challenge, saving the day.† Wouldn’t we all? Well, don’t knock this till you’ve tried it. You’re writing along, and you’ve come to the place where your villain needs to act in some evil way. Your virtual online writing coach has urged you to be sure he has proper motivation. What does this mean? He can’t be bad, do bad, cause trouble just because he’s the bad guy, so what’s made him this way? What’s behind it? You have to know before you have him do whatever it is he’s about to do. Take His Place â€Å"But I’m not a villain!† you say. â€Å"I’m no Dr. Moriarty or Dracula or Simon Legree.† Yes, you are. You have your days. You’ve learned to control yourself, or maybe you’re a person of faith and have found control outside yourself. But you know your true nature, your old nature. We novelists need to become our characters, from young to old, male to female, blue-collar worker to executive, and illiterate to educated. That’s part of the fun of it. Now take that further. When a friend takes credit for something you accomplished, what’s your first private thought? You get over it, I know. You probably say nothing and let it pass for the sake of the relationship, and that’s great. But dwell on that initial visceral reaction a moment. Someone you know well and love and trust lies to you, and there’s no question about it. You’re offended, hurt- crushed really. In fact, you’re infuriated. You bite your tongue because you’re a mature adult. Maybe when you cool down you’ll rationally confront the lie and get to the bottom of it. But for now, entertain that immediate first reaction. Where was your heart and mind then? I’m not telling you to become mean, rotten, and nasty when we’re all supposed to have grown out of that kind of thing by now. But I am telling you to tap into your dark side long enough to know what makes a good villain tick. What Makes a Good Villain? Villains are real people to whom terrible things have happened. Maybe in childhood, maybe in adolescence, maybe later. At some point, rather than learning and growing, their maturation process stunted and stalled. Roots of bitterness and anger sprang up in them. On the surface they may have many, if not most, of the same attractive qualities of your hero. But just beneath the surface fester the qualities you can access in yourself if you allow yourself to. While this may explain the reasons for your villain’s actions, it doesn’t excuse or forgive them. He’s still evil, and he must still be brought to justice. But giving him motivation will make him more than a cardboard cutout. So conjure a backstory for your villain. Make him real and believable and credible- even attractive in many ways. And while you’re writing your story, see how many boxes you can check off on this list of characteristics that pertain to your villain. The more that apply, the more successful your novel is likely to be. Because the more worthy his opponent, the more heroic your hero will appear. Villain Characteristics Checklist: He’s convinced he’s the good guy He has many likeable qualities He’s a worthy enough opponent to make your hero look good You (and your reader) like when he’s on stage He’s clever and accomplished enough that people must lend him begrudging respect He can’t be a fool or a bumbler He has many of the same characteristics of the hero, but they’re misdirected He should occasionally be kind, and not just for show He can be merciless, even to the innocent He’s persuasive He’ll stop at nothing to get what he wants He’s proud He’s deceitful He’s jealous, especially of the hero He’s vengeful Need help writing your novel?Click here to download my ultimate 12-step guide. What would you add to this list of what makes a good villain? Tell me in Comments below.

National Intelligence Estimate (NIE) Policy Simulation Paper Essay

National Intelligence Estimate (NIE) Policy Simulation Paper -Counterintelligence - Essay Example In this battle which has social, budgetary, political, and political and also military sizes – the potential outcomes of counterintelligence disappointments could be quick and destroying, putting in risk our countrys crucial data, base, military powers and an extensive variety of US investment, innovations and faculty far and wide (Goldman, 17-23). In 1978, an arrangement of informal trade visits between US atomic weapons specialists and their Peoples Republic of China (PRC) partners started. The PRC authorities endeavoured to grow close associations with specific US specialists. Over the consequent 23 years, as a consequence of this trade, the PRC made real strides in the improvement of atomic weapons, including the neutron shell. Starting in 1998, US media sources started reporting about continuous examinations of four instances of suspected Chinese surveillance against the United States going over to the 1980s. The most genuine case included Chinas claimed securing of key data about our countrys most developed (The Central Intelligence Agency). US atomic warhead, the W-88, and additionally genuine security breaks at the Department of Energys (DOE) Los Alamos Laboratory between 1984 and 1988 Early in 1998, Congressional center turned to US satellite fares to China. A US Department of Defence grouped report reasoned that researchers from Hughes and Loral Space and Communications, included in concentrating on the 1996 accident of a Chinese rocket propelling a Loral satellite, gave experimental skill to China that prominently enhanced the dependability of Chinas rocket propel capabilities. After this data was distributed in the US media, an extraordinary House Select Committee and various Senate panels researched US innovation exchange approach regarding China. The effect was the arrival of the Report of the Select Committee on U.s. National Security and Military/commercial Concerns

Hybrid Cultural Object Essay Example | Topics and Well Written Essays - 1250 words

Hybrid Cultural Object - Essay Example Language entails the sharing of ideas and thoughts from one person to the other. The culture of the language has been evolving from one community to another. The evolving nature of language has led to the object of communication being improved to a level of being termed as a hybrid language. The most common element of this tool is the use of signs and symbols to communicate. The use of signs has developed significantly due to the interaction of human beings from different regions around the globe. The pressure to find out an aspect of sign language that would unify different cultures in the region have led to the scientific study of signs; semiotics. Signs having been developing, and this study helps the human diversity to know how they are supposed to behave in a particular situation without there being a second person to engage in the verbal communication (Ipsen). The hybrid nature of communication in this state is that signs are being developed into a global unified way that every community get aligned to the communication base to one another. In other words, the signs developed are familiar to everyone around the globe. The global awareness and unity in communication have been established by the fact that there is an organised system that the signs follow. Since there is an organised way of communicating through the use of signs, every stakeholder in the signs language will follow the laid down system. The following of the laid down rules and regulations help different cultural groups to have a unified sign that helps in communication. When a person at one corner of the world sees an image or a picture representing, a situation will be able to give the meaning from the picture and interpret it. Therefore, a person can analyse the situation and act accordingly. It is the globally accepted picture that will help the reader to communicate accordingly in that situation. Signs as used in communication changes

Sucrose Synthase Key Enzyme In Sucrose Metabolism Biology Essay

Sucrose Synthase Key Enzyme In Sucrose Metabolism Biology Essay Sucrose synthase is a key enzyme in sucrose metabolism. Sucrose metabolism is required by the plant to form carbon required for various processes in the plant such as respiration, starch and cell wall formation. The enzyme is encoded by a small multigene family where most plants have at least two isoforms of the enzyme. The kinetics of sucrose synthase show that different Km values and ratios of sucrose breakdown exist for the enzyme. The methods of extracting, assaying and purifying the enzyme are shown in the enzyme characteristics. Factors such as pH, addition of different buffers, metal ions, fungal volatiles as well as environmental factors such as anoxia have all been shown to affect sucrose synthase activity. The enzymes protein sequences have been phylogenetically divided up into six main groups using clustalw. Sucrose synthase is normally present in the cytoplasm but the availability of sucrose in the chloroplast and its ability to use ADP as a substrate would indicate that the enzyme may be able to act in the chloroplast as well as the cytoplasm. Sucrose synthase is an important enzyme in sucrose metabolism in plants cells. (Persia et al., 2008) The main route of entry of carbon from sucrose is commonly known to be sucrose synthase. (Bieniawska et al., 2007) This carbon is used for respiration and in the synthesis of cell wall polymers and starch. (Persia et al., 2008) The main form of reduced carbon in plants is sucrose. It is used to support growth and synthesis of reserve materials e.g. starch in heterotrophic sink tissues. (Matic et al., 2004) The UDP-glucose supplied by sucrose synthase is used for cell wall biosynthesis while working with the cellulose synthase complex. (Baud, Vaultier and Rochat, 2004) In most fruit tissues, an increase in sucrose synthase activity is alongside with sucrose accumulation. This would suggest that sucrose synthase plays a physiologically important role. (Islam, Matsui and Yoshida, 1996) Carbohydrates are transported from photosynthetic source tissues to sink tissues in the form of sucrose . The consequent cleavage of sucrose in the sink tissues is the first step for its use in various metabolic pathways. The sugar is cleaved in vivo by either sucrose synthase (Sus) or by invertase. Invertase catalyses an irreversible reaction where sucrose is cleaved into glucose and fructose (Matic et al., 2004) while sucrose synthase catalyses the reversible conversion of sucrose and uridine-diphosphate (UDP) into uridine-diphosphoglucose and fructose. (Hirose, Scofield and Terao, 2008) (Hardin and Huber, 2004) These enzymes play a crucial role in plant growth and development. (Abid et al., 2009) Sucrose Synthase is cytosolic (Ã…  ebkovà ¡ et al., 1995) and has been characterized in many different plant species such as maize (Hardin and Huber, 2004), rice (Odegard, Liu and Lumen, 1996) and sugarcane (Schà ¤fer, Rohwer and Botha (2005)). Its activity has been studied in many plant organs such as roots, leaves and seeds. (Ã…  ebkovà ¡ et al., 1995) For trees, cellulose biosynthesis is a highly regulated process in which carbon is permanently placed in their primary and secondary cell walls. Sucrose is the main carbon source for cellulose synthesis. The stem is made up of extremely active sink cells which utilise sucrose for cellulose synthesis. Sucrose synthase is the main sucrolytic enzyme in these cells that catalyzes the reversible conversion of sucrose into fructose and UDP-glucose which is needed for cellulose biosynthesis. (Joshi, Bhandari and Ranjan, 2004) It also plays an important role providing adequate sugar supply during anoxic stress. It has been shown that during anoxic germination of rice, sucrose synthase activity was enhanced whereas the activity of invertase was depressed. This would indicate that sucrose synthase is the enzyme predominantly responsible for sucrose breakdown during anoxia. (Joshi, Bhandari and Ranjan, 2004) Fig 1: Diagram of the cleavage and synthesis reaction of sucrose synthase (Rà ¶mer et al., 2004) Different isoforms of the gene are present in most plants. In the case of maize, two non-allelic genes were discovered for sucrose synthase but more investigation lead to the discovery of a third. At least three genes for sucrose synthase have been discovered in rice where the genes show differences in expression between tissues. RSus1 is expressed in root phloem while RSus2 is expressed in leaf phloem. (Schà ¤fer, Rohwer and Botha, 2005) When examining the different isoforms at an amino acid level it is appears that there is less homology between different sucrose synthase genes in a species than when the gene is compared to its corresponding gene in another species. In the case of maize, there is 75% homology between the SS1 gene and SS2 gene of maize but there is 90% homology between rice RSus1 and maize SS2 genes. In sugarcane, the SS1 gene is 97% identical at the amino acid level to maize SS1 gene. (Lingle and Dyer, 2001) Nolte and Koch (1993) undertook a study to determine whether sucrose synthase was localized to certain part of the vascular strand. It is well known that sucrose synthase is present in vascular bundles for example in transgenic tobacco plants phloem specific expression of a maize sucrose synthase gene has been observed. Their study, using immunohistochemistry, found that sucrose synthase was restricted to the cytoplasm of companion cells of the phloem and did not appear to be present in other organelles of the plant. (Nolte and Koch, 1993) The molecular mass of sucrose synthase can be determined by gel filtration. Sucrose synthase elutes from the column with a Kav value of 0.17844 which when using a calibration curve correlates to a molecular mass of 362kDa. Using SDS-PAGE gradient gel the molecular mass of each subunit can be estimated at 92kDa. This can conclude that sucrose synthase is a tetrameric enzyme with a molecular mass of 360kDa and four identical subunits of 90kDa. (Hardin and Huber, 2004) (Elling and Kula, 1993) It can associate with membranes and the actin cytoskeleton where its activity is known to be involved with cellulose synthesis. It does this by channelling uridine-diphosglucose to the growing glucan chain by the enzyme cellulose synthase. (Hardin and Huber, 2004) Analysis of Sucrose Synthase Gene Family: From the results of species examined to date, it is shown that sucrose synthase is encoded by a small multigene family. (Bieniawska et al, 2007) Most species of plants have at least two isoforms of sucrose synthase. These isoforms usually have comparable biochemical properties and highly homologous amino acid sequences. (Wen et al., 2010) Further analysis of transgenic and mutant crop plants show certain isoforms of sucrose synthase have specific functions in the plant. The rug4 mutation of pea removes the SUS1 isoform but has no effect on SUS2 or SUS3. This would indicate that these two isoforms are not able to make up for the loss of SUS1 in the seed or root nodule. It is clear that the loss of different isoforms affect the plant in certain ways. Loss of the SH1 isoform in maize has different outcomes from the loss of SUS1 isoform. SH1 is required for normal cell wall formation during endosperm development while both isoforms are needed for wild-type rates of starch synthesis. Why different isoforms have different functions is unclear. The same functions can be carried out in the cell by different isoforms but can occur in distinct cell types, developmental periods or environmental conditions. It is likely that different isoforms could have non-overlapping, particular functions in the same cell. (Bieniawska et al., 2007) It is difficult to decide on the precise roles of the genes in sucrose synthase gene family when there is not enough information in existence. Although there is some information available on some of the isoforms and theyre functions in the plant, no analysis of the functions of the gene family has been carried out. The model plant Arabidopsis is ideal for carrying out such an analysis. Six sucrose synthase genes are in the Arabidopsis genome. Based on comparisons of the amino acid sequences the isoforms they encode can be divided into three distinct pair groups. The isoforms SUS1 and SUS4 are 89% identical to each other but have less than 68% similar amino acid sequences to other isoforms. Similarly, SUS2 and SUS3 are 74% identical to other isoforms and are 67% less identical to the other forms of enzyme. SUS5 and SUS6 are 585 identical to each other but have less 48% similarity to the other isoforms. When examining other dicotyledonous species it appears that at least two of the thr ee pairs of isoforms are present. When phylogenetic analysis was carried out, it showed that the isoforms AtSUS1 and AtSUS4 are related to pairs of isoforms from pea (Fabacae), carrot (Umbelliferae) and potato (Solanacae). A pair of isoforms from Craterostigma plantagineum (Scrophulariacae) is closely related to the pair of isoforms AtSUS2 and AtSUS3 in the Arabidopsis. The pair AtSUS5 and AtSUS6 is related strongly to a pair of genes from rice. This evidence shows that it is unlikely that the three pairs of isoforms in Arabidopsis are as a result of gene duplication events. It is possible that each isoform has an exact function preserved in a wide range of plants. The members of Arabidopsis gene family are strongly differentially expressed in different organs of the plant through its development and in response to external stimuli e.g. environmental stress. This is seen in gene families of other plants studied. (Bieniawska et al., 2007) Fruit quality is determined by the type and quality of sugars present. A study of the sucrose synthase-encoding gene from the muskmelon fruit was carried out to evaluate how to genetically improve the quality of the fruit. This is done by finding the sugar components in fruit, to identify the enzymes involved in sugar metabolism and distinguish the relationship between sugar accumulation and the activities of related enzymes. It is thought that sucrose synthase is the enzyme involved in metabolising sucrose in developing muskmelon fruit. To examine this, a full length cDNA strand encoding sucrose synthase was extracted from a muskmelon fruit by RT-PCR and RACE and identified as CmSS1. Real time PCR analysis showed that CmSS1 expression changed in among different tissues of the plant e.g. root, stem, leaf. It showed that the mRNA levels are highest in the root and lowest in mature fruit. Fig 2: The patterns of CmSS1 transcript abundance in the different tissues of the muskmelon plant. These results were found using quantitative real-time PCR analysis of total RNA prepared from the root, stem, leaf, flower and mature fruit of muskmelon. During fruit development and ripening it was shown that CmSS1 mRNA was at its maximum level at five days after pollination and decreased steadily during fruit development until it reached its minimum level of maturity. This was discovered using again real-time RT-PCR analysis of mesocarp tissues from five days of pollination to ripening. Fig 3: This graph depicts the patterns of CmSS1 transcript abundance in developing muskmelon fruits found by using quantitative real-time PCR analysis of total RNA prepared from muskmelon. (Wen et al., 2010) The sugar content and SS activity were analysed to show the functions of CmSS1 in regulating fruit quality. It showed that very low concentrations of sucrose are present in young and unripe muskmelons. Between 20 and 30 days after pollination there is a massive rise in the amount of sucrose in the fruit. Sucrose synthase activity increased in the direction of sucrose synthesis and decreased in the direction of sucrose cleavage through fruit development. (Wen et al., 2010) Fig 4: The depiction of sucrose content and sucrose synthase activity during muskmelon fruit development. The first chart shows sucrose content during fruit development. The second shows sucrose activity in the sucrose synthesis direction and the third shows sucrose cleavage direction during muskmelon fruit development. (Wen et al., 2010) Enzyme Kinetics of Sucrose Synthase: An investigation was carried out by Schà ¶fer et al. to the find the properties of three sucrose synthase isoforms present in sugarcane. Kinetic analysis indicated that the three sucrose synthase genes in sugarcane are different isoforms, with major differences in Km values and the ratios of sucrose breakdown synthesis. The kinetic characteristics of the SuSyA and SuSyB isoforms, both expressed in the leaf roll, differ greatly. It was found that SuSyA has almost three times higher affinity for sucrose than the SuSyB isoform whereas SuSyB has a much greater affinity for UDP than SuSyA. Based on the differences in their kinetic properties it can be concluded that SuSyB and SuSyC are different isoforms of sucrose synthase. SuSyC has roughly ten times higher affinity for UDP compared to the other two isoforms. (Schà ¤fer et al., 2005) Fig 5: The graph shows the Lineweaver-Burk plot of 1/v against 1/S for the isoforms SuSys A, B and c where UDP was the variable substrate. The concentration of sucrose was kept constant at 320nM. The Km values were determined from the non-linear fit of the data to the Michaelis-Menten equation. (Schà ¤fer et al., 2005) When examining sucrose synthase in soybean nodules Morell and Copeland (1985) found the kinetic constants of UDP, UDPglucose, sucrose and fructose by fitting the data to the following two equations: 1. v = VA/KiaKh + KhA + KhB + AB 2. v = VA/Ka + A + A/Ki The kinetic constants for ADP, CDP and ADPglucose were found using non linear regression analysis of initial velocity data. Fig 6: Graph showing the effect of sucrose concentration on the cleavage activity of sucrose synthase in soybean nodule. The lines show the fit of data to equation 1. The reaction mixture were composed of 20 µmol Hepes-KOH buffer (pH 7.5) 2 µmol UDP, 1.5 µmol NAD, 25 µg UDPglucose dehydrogenase. Each symbol represents a different concentration of sucrose. The dark circle shows 3.2 µM, the clear circle shows 4 µM, the dark triangle shows 6.25 µM, 10 µM is shown by the clear triangle and the dark square depicts 20 µM. In the cleavage and synthesis direction standard Michaelis-Menten kinetics are observed. The variation of concentration of sucrose at different concentrations of UDP gave an intersecting pattern of linear double reciprocal plots. (Morrell and Copeland, 1985) Parameter Value V (U/mg protein) 13.3 ±2.0 Km sucrose (mM) 31.3 ±7.1 Ki sucrose (mM) 31.9 ±13.1 Km UDP (mM) 0.005 ±0.002 Ki UDP (mM) 0.005 ±0.001 Fig 7: Table showing the kinetic parameters for the cleavage reaction of sucrose synthase in soybean nodules. (Morrell and Copeland, 1985) Fig 8: The graph depicting the effect of UDPglucose concentration on the synthesis reaction of sucrose synthase activity in soybean nodules. The reaction mixtures contained 20 µmol Hepes-KOH buffer, 15  µmol fructose, 5 µmol MgCl2, 0.4  µmol P-enolpyruvate, 0.15  µmol NADH, 20 µmol KCl, 25 µg pyruvate kinase 25 µg lactate dehydrogenase and the required amount of enzyme. As in the previous graph, the amount of UDPglucose was varied in the presence of 2.5mM (dark circle), 3.2mM (clear circle), 4mM (dark triangle), 5mM (clear triangle) and 8mM (dark square) fructose. The results on the graph are representing the fit of data to equation 1. When the concentration of UDPglucose was varied at the concentrations of fructose in the graph, an intersecting pattern of linear double reciprocal plots was seen. From fitting the data from the graph to equation 1, it is noted that substrate inhibition would have occurred at a concentration greater than 15mM fructose. Parameter Value V (U/mg protein) 14.3 ±1.2 Km fructose (mM) 3.7 ±0.8 Ki fructose (mM) 19.6 ±9.9 Km UDPglucose (mM) 0.012 ±0.006 Ki UDPglucose (mM) 0.064 ±0.014 Fig 9: table showing the kinetic results by fitting the figures from the graph to equation 1. When partially purified SuSyA, SuSyB and SuSyC were blotted to a nitrocellulose filter the results showed that all three isoforms are approximately 94kDa. (Schà ¤fer et al., 2005) The would correlate to the findings of Hardin et al and Lothar et al who stated that sucrose synthase is tetrameric enzyme made up of four 90kDa subunits. Fig 10: Immunoblot of sugarcane SuSy. A crude extract of protein from leaf roll was loaded into lane 2 while partially purified isoforms of SuSyA, SuSyB and SuSyC were loaded to lane 3, 4 and 5. The molecular weight ladder was used to identify the bands see in each lane. (Schà ¤fer et al., 2005) Characteristics of Sucrose Synthase: Extraction of Protein: The method for extracting protein from the leaves of maize (Zea mays), rice (Oryza sativa) and tobacco was done as follows: 1-3g of leaves was ground in liquid nitrogen and the powder was mixed in the ratio 1:2 with extraction buffer. The buffer was made up of 0.1M tris-HCl, pH 8, 10mM DTT and 1% polyvinylpolypyrrolidone. The samples were then incubated on ice for 15 minutes and then centrifuged at 1,000g for 10 minutes at 4oC. The pellet was then removed and the supernatant was re centrifuged at 100,000g for one hour at 4oC. After this final centrifugation, the pellet and supernatant which contained the soluble proteins was resuspended in sample buffer for electrophoresis. (Persia et al., 2008) When extracting protein from rice seeds, a similar procedure is followed. Seeds weighing roughly 50-100mg at various stages of growth were homogenized in 400 µl of extraction buffer and kept at 4oC. The buffer was made up of 50mM Tris-HCl, pH7.5, 1.0mM DTT, 1.0mM EDTA and 2mM PMSF. Ammonium sulphate fractions (30-50% w/v) were precipitated and then resuspended in dialysis buffer made up of 50mM Tris-HCl, pH 8.0, 5mM MgSO4, 5mM 2-mercaptoethanol. This was then dialyzed overnight at 4oC. (Odegard, Liu and De Lumen., 1996) The method for extracting protein from tobacco pollen tubes is slightly different to those mentioned previously. The pollen first was slowly thawed from storage at -20oC and hydrated in a humid chamber overnight. It was then germinated in BK medium and allowed to germinate at 25oC for three hours. After this period had elapsed, the pollen was collected by centrifugation at 1,000g for 5 minutes at 25oC. It was then washed twice with BRB25 buffer which is made up of 25mM HEPES, pH 7.5, 2mM EGTA and 2mM MgCl2 and 15% Suc. After washing, the pollen was resuspended in lysis buffer and lysed on ice using a motor-driven Potter-Elvehk-jem homogenizer. The lysis buffer used was made up of BRB25 buffer along with 2mM dithiothreitol, 1mM phenylmethylsulfonyl fluo ride (PMSF), 10 µL/mL protease inhibitors, 1mM NaN3 and 10% mannitol. After lysis was carried out, the samples were centrifuged at 1,000g for 10 minutes at 4oC. The supernatant was centrifuged again at 4oC for 45 minutes at 100,000g over a 20% (w/v) Suc cushion. The supernatant was then collected as it contained the soluble protein fraction. (Persia et al., 2008) Enzyme Assays: After extracting protein, the sucrose synthase activity in sugarbeets was found using a spectrophotometric end point assay. The activity of the enzyme was monitored as fructose formed at 35oC. This was carried out in a solution that contained 250mM sucrose, 2mM UDP and 100mM MES. The control was carried out by assaying for activity in the absence of UDP. The total protein concentration was determined using the Bradford method where bovine serum albumin was the standard. (Klotz and Haagenson., 2008) When assaying for protein from rice, the Bradford method was followed to determine protein concentration as was done in Klotz et al. 40mg of protein was used per assay. The assay was carried out in 20mM MES pH 6.4, 200mM sucrose and 4mM UDP for 15 minutes at 30oC. The reaction was stopped by boiling for 2 minutes and the fructose levels were measured. The control tubes did not contain UDP. (Odegard, Liu and De Lumen., 1996) When examining the effect of sucrose synthase on carbon partitioni ng a similar method was followed for assaying the protein. Sucrose synthase was assayed in the direction of sucrose breakdown using 50 µl poplar plant extract. The tetrazolium blue assay was followed to determine the amount of free fructose. As in previously mentioned assays, the absence of UDP in the assay acted as a control. The total protein content was found by employing the Bradford (Bio-Rad) protein assay. (Coleman, Yan and Mansfield., 2009) A similar method was followed for carrying out an assay for the enzyme on tomato tissue. The reaction mixtures contained 50mM Hepes-NaOH buffer, 15mM MgCl2, 25mM fructose and 25mM UDP glucose. This was incubated at 37oC for 30 minutes and was terminated with the addition of 70 µl of 30% KOH. The enzyme blanks were terminated with the addition of KOH at 0 minutes. The tubes were then kept at 100oC for 10 minutes to destroy any fructose. The soluble protein content was determined using the Lowry method whereby bovine serum albumin was th e standard. (Islam, Matsui and Yoshida., 1996) Alkaline copper solution is added to each tube and allowed to stand at room temperature for roughly 30 minutes. Dilute folate reagent is then added to each tube rapidly and after 30 minutes the absorbance is read at 750nm. (Lowry et al., 1951) The results were measured as  µmole of sucrose per minute per mg protein. (Islam et al., 1996) When assaying for sucrose synthase in the cleavage direction Rà ¶mer et al used recombinant SuSy1 gene from potato. In a volume of 100 µl HEPES buffer with a concentration of 200mM and pH 7.6 recombinant sucrose synthase was incubated along with 2mM UDP and 500mM sucrose for ten minutes at 30oC. HPLC analysis was used for the formulation of UDP-glucose. The Bradford assay was used to determine protein concentrations as was carried out by Klotz et al and Coleman et al. The activity of the enzyme was also tested with the nucleoside diphosphates dTDP, CDP, ADP and GDP at 2mM. For assaying recombinant e nzyme in the synthesis direction a similar method was followed as when assaying for standard enzyme. Recombinant sucrose synthase was incubated in a total volume of 100 µl HEPES buffer where this time the pH was 8.0 and the concentration was as in cleavage direction of 200mM. 1mM UDP-Glc and 20mM D-fructose was also added to the mixture and it was incubated for five minutes at 30oC. The reaction was heated to 95oC for five minutes and HPLC analysis was used to establish the formation of UDP. The sucrose synthase activity was also tested using dTDP-Glc, CDP-Glc and ADP-Glc. (Rà ¶mer et al., 2004) Purification of Protein: After extraction of the protein from the crude extract, purification can be carried out. This can be done in a number of ways such as Batch adsorption with Sephadex A50, Anion exchange chromatography and Gelfiltration. SDS-PAGE can be carried out after purification to check the purity of the protein sample. The Sephadex A50 gel is loaded into a glass funnel and washed twice with deionised water. The gel was then washed twice with 300ml standard buffer. The protein sample was loaded to the gel and slowly sucked through the gel for 30 minutes. The gel bed was then washed with 300ml standard buffer and then with 300ml standard buffer containing 100mM KCl. The last washing step contained 300mM KCl. 200ml of the first salt preparation was concentrated to 40-50ml by using a cross-flow ultrafiltration module with YM 30 ultrafiltration membrane that had been pretreated with 55 PEG 4000 solution. This was done to prevent the enzyme sticking to the membrane. In anion exchange chromatography a Sepharose Q column was first equilibrated with 300ml Hepes buffer. This was made up of 200mM pH 8 with 50mM KCl. 70-80mg of protein sample was loaded and the elution was started using two different salt gradients. To prevent enzyme inactivation after elution all the fractions were titrated back to pH 7.2. All fractions that contained enzyme activity were pooled and concentrated by using ultrafiltration. Gelfiltration experiments are carried out on a prepacked HiLoad 16/60 Superdex 200 prep grade column that was connected to FPLC equipment. Four samples containing 2mg of protein were loaded and eluted with a flow rate of 1 ml min-1. The fractions were then pooled and stored at -20oC in 500 µl aliquots. (Elling and Kula., 1993) To determine the purity of the protein, SDS-PAGE is carried out. This is done by loading 100 µg of protein samples to a 125 SDS-polyacrylamide slab gel that was overlaid with stacking gel. The electrophoresis was carried out at 4oC and at 40V for 16 hours a nd followed by 200V for one hour. Coomassie blue R 250 was used to stain the gel followed by destaining. (Kumutha et al., 2008) Factors that affect Sucrose Synthase Activity: Ã…  ebkovà ¡ et al (1995) stated that sucrose synthase has two different pHs for optimal activity. In the cleavage direction it was found that most enzyme activity was observed between pH 6.0 and 8.5 at temperatures between 50 to 55oC. In the synthesis direction, a pH between 8.5 to 9.5 and a temperature of 35oC was optimal for enzyme activity. (Ã…  ebkovà ¡ et al., 1995) This would correlate with the findings of Morell and Copeland (1985) who found that optimal activity of the enzyme in soybean was at pH 6 in the cleavage direction and at a pH of 9.5, sucrose synthase activity in the synthesis direction was at its highest. It was also found that at a pH of 7.5 the cleavage and synthesis activities were their highest. (Morrell and Copeland., 1985) Elling and Kula (1995) examined the effect of buffers TES-NaOH, MOPS-NaOH, TEA-NaOH and Tris-HCl on the pH optimum of sucrose synthase activity. These were determined using UDP and TDP as substrates for the reaction. They found that the e nzyme had its highest activity in Hepes-NaOH buffer. When MOPS-NaOH and TES-NaOH buffer was used, only 60-80% activity was noted. (Elling and Kula 1995) It was also found that the velocity of the reaction could be increased by increasing the temperature where optimal activity was seen between 50 and 60oC. Xu at al (1989) reported that potato and bean are also able to withstand these high temperatures. However once the temperature goes above 60oC enzyme activity starts to decreased rapidly and was destroyed once the temperature reached 70oC. (Xu et al., 1989) The cleavage of sucrose by the sucrose synthase enzyme was investigated to find the rate of cleavage reaction using different nucleosidediphosphates as cosubstrates. They found that the rate of reaction was UDP>TDP>ADP>CDP>GDP. Echt and Chourey (1985) found similar results when examining nucleotide specificity. They found that substrate specificity for SS1 and SS2 were UDP>TDP>ADP>CDP>UTP where each substrate was at a concentrat ion of 4mM. (Echt and Chourey 1985) Low levels of heavy metal ions such as mercurate inhibited cleavage activity of the enzyme. This would lead to the assumption that sulfhydryl groups are involved in the catalytic process. It is also inhibited by Tris-HCl and by small concentrations of MgCl2 and MnCl2. (Ã…  ebkovà ¡ et al., 1995) Cations were shown by Elling and Kula (1995) to have a slight influence on enzyme activity. The activity was lessened slightly (10%) by the presence of 1mM Mn2+ and Mg2+ ions with UDP. The enzyme is completely inactivated in the presence of 1mM Cu2+ or Fe2+. (Elling and Kula., 1993) A recent study was undertaken to examine the effects of volatile emissions on carbohydrate metabolism. Studies on this area have taken place before but it is usually examining the results of physical contact between the host plant and the microbe. No work has taken place until now on the effect on the plant in the absence of physical contact. Many microbes such as Pseudomonas spp, Strepomyces spp, Penicillin spp and a selection of truffles produce ethylene. This gaseous plant hormone plays an important role in many aspects of plant growth and development such as seed germination, root hair initiation, fruit ripening and starch accumulation. In the work of Ezquer et al (2010), the possible effects of volatiles released from gram-negative bacteria, gram-positive bacteria and fungi on starch metabolism was studied. The results showed that the volatile compounds released by microbes promoted high levels of starch accumulation in mono- and dicotyledonous plants. It also revealed fungal vo latiles (FVs) promoted massive changes in expression of genes involved in many important processes in plant such as metabolism of carbohydrates, amino acids, sulphur and lipids, energy production, protein translation and stability, cell wall biosynthesis and photosynthesis. However no changes were noted in the expression in some of the genes that coded for proteins involved in starch and sucrose metabolism such as plastidial hexokinase, plastidial phosphoglucose isomerase, plastidial adenylate kinase, alkaline invertase and UDPglucose (UDPG) pyrophosphorylase. It was found in the study that FVs strongly upregulate the expression of Sucrose Synthase in potato leaves. The plants were cultured in the presence and absence of FVs emitted by A. Alternata. This caused a massive enhancement of expression of Sus4 isoform. A 29.4- and 31.63-fold increase was observed in expression when the plants were cultured in the presence and absence of sucrose. This isoform of the enzyme controls the accumulation of ADPG, UDPG and starch in potato source leaves and tubers. Analyses of the intracellular amounts of starch and nucleotide-sugars in the leaves of the plant show a positive correlation between patterns of enzyme activity and starch, UDPG and ADPG amounts. This was noted when the leaves were cultured in the presence and absence of FVs. Western blot analyses and quantitative RT-PCR confirmed also the increase in expression. (Xu et al., 1989) Environmental Factors affecting Sucrose Synthase Activity: Anoxia: Waterlogging is where oxygen supply is blocked to root leading a severe decrease in the amount of oxygen available to the plant. This leads to inhibition of root respiration that causes a major decline in energy of root cells affecting vital metabolic processes of the plant. This is restriction of oxygen supply is known as anoxia. The presence of glucose in an anoxic incubation medium drastically decreases meristem death and studies have shown that sucrose synthase is the enzyme mainly responsible for sucrose breakdown under anoxia. (Kumutha et al., 2008) The increase in glycolytic demands caused by these demands is the cause of increased sucrose synthase expression. This has been demonstrated in many plant species e.g. sucrose synthase gene is induced in wheat and in rice when oxygen levels are low. (Ricard et al., 1998) Harada et al (2005) also found an increase in sucrose synthase activity in pondweed turins while under anoxia. (Harada et al., 2005) Klotz and Haagenson (2008) foun d that sugarbeet contained two genes for sucrose synthase activity-SBSS1 and SBSS2. They demonstrated that anaerobic conditions caused a large increase in the transcription levels of SBSS1 and a quick increase and succeeding decline in SBSS2 transcription levels. However this did not correlate with a significant increase in sucrose synthase enzyme activity. A 23% increase in sucrose synthase activity was noted after initiation of anaerobic conditions but otherwise the activity of the enzyme did not differ greatly to that of the controls. (Klotz and Haagenson., 2008) Fig 11: The graph outlines the different rates of sucrose synthase activity in the con