Paper #1

Molecular Machines for Developing Countries:
Malaria and G6PDMalaria is the most common, widespread, and destructive infectious disease in the developing world; it is a significant public health risk to people inhabiting and visiting developing countries. According to the Centers for Disease Control and Prevention website, there are approximately 350-500 million cases of malaria recorded each year; more than one million of these people die (“Malaria”). Malaria is the result of poverty in the developing world; regions of the world where malaria is prominent do not have sufficient medical resources. The people of these regions also have unsanitary living conditions and are surrounded by destitution. The regions of the world that experience the most malarial outbreaks are tropical and subtropical regions, such as areas in the Americas, Africa, and Asia. Children of Sub-Saharan Africa constitute the majority of the death-toll; malaria parasites also maliciously affect the health of pregnant women.

Some worrisome symptoms and bodily defects induced by malaria parasites include: fever, joint pains, spleen and liver enlargements, and hemoglobinuria. A classical symptom of malaria is fever, caused by multiple bursts of merozoites exploding their host red blood cells in the bloodstream. A malaria-infected individual will also experience cycles of chills and rigor that differ in length depending on which strain the person has contracted. Some malaria symptoms and their causes are still poorly understood, which provides incentive for scientists, researchers, and sponsors to unearth and fund malaria prevention methods.

Plasmodium protozoan parasites cause malaria. These parasites are unicellular microbes that symbiotically rely on their host species. Anopheles female mosquitoes feed on the blood of a malaria-infected person when young, thus becoming infected with the parasites as well. Therefore, infectious malaria parasites are spread through vector transmission from a mosquito to a human. Plasmodium sporozoites travel in the saliva of Anopheles females into the bloodstream of a human. These sporozoites rapidly progress into the first of their two pathways. They infect the hepatocytes of the liver within the 30 minutes following their invasion of the bloodstream; this is the exoerythrocytic pathway. The sporozoites asexually proliferate for one to two weeks before their mass-produced merozoites break free of the host cells, traveling through the blood to claim red blood cells as their new hosts; this is the erythrocytic phase of the parasite’s life cycle. There are four malaria parasites that are infamous for infecting humans. These parasites are: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae; P. falciparum is the most severe form of the disease.

However, molecular studies and research provide hope for malaria prevention, especially of the P. falciparum strain. P. J. Mason et al. believe that human red blood cell glucose-6-phosphate dehydrogenase (G6PD) somehow relates to the G6PD of P. falciparum. They find that the C-terminal sequence of human red blood cell (HRBC) G6PD is homologous to the N-terminal of P. falciparum G6PD (30). So the structures of the C-terminal sequence of HRBC G6PD and the N-terminal of P. falciparum G6PD could share similar structures, but have different functions, or they could have corresponding or related locations within their proteins. The “C-terminal [is defined as] the end of a protein (either the polymerases involved in the process of transcription or the polypeptide product of the DNA) corresponding to the 3’end of the coding sequence for that protein” (“Griffiths”). The “N-terminal [is defined as] the end of a protein (either the RNA polymerases involved in the process of transcription or the polypeptide product of the DNA) corresponding to the 5’end of the coding sequence” (“Griffiths”). A peptide sequence is written from the left-hand N-terminal to the C-terminal. Since these opposite sequences are structurally similar, the parasite could act conversely to the human red blood cells, moving from the 3’ to the 5’ end instead of from the 5’ to the 3’ end when the sequence is being read. This possibly reversed process is a definite avenue of research that would involve studying the effects of the varying processes.

G6PD is an enzyme that catalyzes the first step in the pentose phosphate pathway. In this step, NADP⁺ is transformed into NADPH due to the conversion of glucose-6-phostphate into 6-phosphoglucono- δ-lactone by G6PD. Cells need NADPH in order to protect themselves from oxidative stress. This stress comes from a variety of triggers. Oxidants, which are potent oxygen compounds, build up when an individual contracts a fever or when he or she ingests certain medications or foods, such as fava beans. G6PD also accounts for the normal life span for red blood cells. The following diagram, from GenomeNet, provides a summary of the first step in the pentose phosphate pathway (“KEGG REACTION”):

The pentose phosphate pathway is metabolic; it provides the reducing energy for cells by maintaining NADPH levels. NADPH manages glutathione level in cells, which is the mechanism that protects red blood cells from oxidative damage.

G6PD can be homodimeric or tetradimeric; the latter of which is found in humans. It has eight chains that resemble ribbons. These ribbons contain NADP molecules embedded within the proteins. G6PD is a multimer; generally, eight NADP molecules are found within a large protein. The following picture is a G6PD tetramer that was found in an article on ScienceDirect.com (Shannon et al.):

G6PD deficiency is a disease where red blood cells do not have the G6PD enzyme. Oxidative stress triggers—foods, illnesses, medications—cause individuals to suffer the symptoms of G6PD. The inheritance of G6PD deficiency is sex-linked; it is transmitted from a healthy-carrier mother to her son, since mostly males display the symptoms of this disease because the gene for G6PD is carried on the X chromosome. Daughters that inherit the trait would be healthy carriers like their mothers. G6PD deficiency is the most common enzyme deficiency disease in the world. There are quite a few advantages and disadvantages to G6PD deficiency. For example, one disadvantage is that hemolytic anemia can result if a G6PD deficient patient undergoes oxidative stress.

G6PD deficiency research would be advantageous to malaria prevention in the developing world because, for unknown reasons, malaria parasites do not survive in G6PD deficient cells. Some other advantages to this deficiency include resistance to cancer, stoke, and cardiovascular disease; resistance to the most morbid form of malaria—P. falciparum—is a major advantage since most of the malaria parasite’s life is spent in either the liver or red blood cells, causing it to easily avoid the immune system so easily and be incredibly problematic to cure or prevent. Furthermore, merozoites can wrap themselves in the lysed liver cells once they leave the liver and move to red blood cells, disguising themselves to avoid attacks from the immune system. The sticky surface proteins of P. falciparum can be shifted between a wide variety of roughly 60 or more types of proteins per parasite to evade recognition by the immune system; parasite populations can thus have endless variations and combinations of these surface proteins. Patients with malaria can experience hemorrhages because of the sticky surface proteins that P. falciparum attaches to red blood cells, causing them to stick to blood vessel walls, halt the merozoites’ circulation, and avoid destruction in the spleen.

The studies of G6PD and its structure and function can be applied to the developing world. Researchers could study G6PD deficiency inheritance more in-depth so that a method of controlling the severity of the deficiency through its genetic expression can be found. This way, someone could conceivably be protected from malaria without experiencing the harsh side-effects of G6PD deficiency. Favism is not as widespread as malaria, but since it is another name for G6PD deficiency, it is another avenue for research and application. Favism seems to be developing as an evolutionarily advantageous genetic trait in regions of the world where malaria is prominent (“What is G6PD Deficiency?”). Since this condition is occurring naturally, it should be further researched as a malaria prevention method.

Malaria is the result of poverty in the developing world, but it also causes poverty and poor economic development. This causes an enormous struggle for inhabitants of developing countries because of their increased struggle for wealth and a significant decrease in their already poor quality of life. Jobs become difficult to create and maintain, and thus the well-being of the inhabitants suffers. Family members that were a significant source of income die, leaving emotionally and financially distraught relatives, and because the disease is so widespread and the economy is hindered, those that are still suffering lack adequate medical care. If more enzymes or pathway variations or mutations are discovered that protect against malaria with few other symptoms and side-effects, the developing world would significantly benefit.

Works Cited

Griffiths, Paul, and Karola Stotz. "Glossary." Representing Genes: Testing Competing Philosophical Analyses of the Gene Concept in Contemporary Molecular Biology. 31 Oct. 2006. University of Pittsburg. 22 Feb. 2008 .

"KEGG REACTION: R00835 R02736." GenomeNet. Feb. 2008. Kyoto University Bioinformatics Center. 22 Feb. 2008 .

"Malaria: Topic Home." Centers for Disease Control and Prevention. 31 Jan. 2008. Dept. of Health and Human Services. 22 Feb. 2008 .

Mason, Philip J., David Stevens, Amalia Diez, Stuart W. Knight, Deborah A. Scopes, and Tom J. Vulliamy. "Human Hexose-6-Phosphate Dehydrogenase (Glucose 1-." Blood Cells, Molecules, and Diseases 25 (1999): 30-37. 22 Feb. 2008 .

Shannon, W N., Sheila Gover, Veronica M. Lam, and Margaret J. Adams. "Human Glucose-6- Phosphate Dehydrogenase: the Crystal Structure Reveals a Structural NADP+ Molecule and Provides Insights Into Enzyme Deficiency." Structure 8 (2000): 293-303. 22 Feb. 2008 .

Bibliography

Adams, John H. "Malaria." Microsoft® Encarta® 2006 [CD]. Redmond, WA: Microsoft Corporation, 2005.

Blandin, Stephanie A., and Elena A. Levashina. "Reverse Genetics Analysis of Antiparasitic Responses in the Malaria Vector, Anopheles gambiae." Innate Immunity. New Jersey: Humana P, 2008. 365-377.

"Blood disease." Encyclopædia Britannica. 2008. Encyclopædia Britannica Online. 10 Feb. 2008. .

Boëte, Christophe, ed. Genetically Modified Mosquitoes for Malaria Control. Georgetown, TX: Landes Bioscience, 2006.

“Glucose-6-phosphate dehydrogenase deficiency." Encyclopædia Britannica. 2008. Encyclopædia Britannica Online. 10 Feb. 2008. 9037085>.

Mattingly, P. F. The Biology of Mosquito-Borne Disease. New York: American Elsevier Company, Inc., 1969.

"Metabolism." Encyclopædia Britannica. 2008. Encyclopædia Britannica Online. 10 Feb. 2008. .

T-W-Fiennes, Richard N. Zoonoses and the Origins and Ecology of Human Disease. New York: Academic P, 1978.

"What is G6PD Deficiency?" G6PD Deficiency Association. 2008. UNIAMO ( Italian Federation for Rare Diseases). 10 Feb. 2008 .

Sorry- the pictures didn't upload with it!

Paper 1

Restriction Enzymes

Since the discovery of restriction enzymes, the world of genetic engineering has become a major player in biotechnology. Genetically altering DNA sequences in prokaryotic and eukaryotic cells has provided hope to a plethora of issues which face mankind today. One of these issues is the buildup of greenhouse gases and the steady increase in global temperature since the industrial revolution of the early 1900s. Restriction enzymes have allowed scientists to isolate favorable traits that are represented in the DNA in plants, which could ultimately lead to these traits to be present in all plants. The way in which restriction enzymes work is invasive enough to successfully cut the desired DNA sequence. However it does not interfere with the replication or translation of the DNA and because of this characteristic it has become a staple of biotechnology research and product development. Although restriction enzymes work on the nano-scale level, the ramifications of harnessing the power of this naturally produced enzyme can provide possible solutions to issues which are on the macro-scale.

The facts, which support the notion that the world is slowly increasing in temperature, are irrefutable. According to research complied by the United State Environmental Protection Agency, the global mean temperature has risen between 1.0 and 1.7^o F since 1850 (6). The temperature increase has a direct relationship with the increase of greenhouse gasses. Scientists have recorded a 36% increase in the amount of carbon dioxide in the atmosphere, whereas methane has seen a 148% increase since pre-industrial times (6). The increase in these two gasses is almost nearly all due to human involvement. The increase in the amount of greenhouse gasses in the future will also exponentially speed up the warming of the Earth. Projections estimate that the Earth will increase its surface temperature between 2 and 11.5^oF by the end of the 21^st century (6). Increases like these would severely cripple the size of the polar ice caps, and, subsequently, ocean levels would rise and then jeopardize the integrity of many major cities around the world.

However, this future scenario is not for certain. Many projections exist for the increase in greenhouse gasses and temperature, but these projections range significantly. These variations between the predictions are due to the unknown impact which humans may have on the environment. On one hand, CO₂ and CH₄ emissions may continue to increase exponentially, and when this is paired with no human effort to slow the temperature increase, the more extreme highs are produced. On the other hand, human involvement is the key to slowing down the global climate change. The human factors which would affect global warming include the following: stricter regulations on greenhouse gas emission, reducing consumption of fossil fuels, and using any form of technological advances to decrease the amount of greenhouse gasses in the air. These restrictions and technological advances are occurring all of the time, and one recent breakthrough is the use of restriction enzymes to genetically alter plants.

Restriction enzymes are like a pair of very accurate scissors on the cellular level. The enzymes are used to cut strands of DNA in a very particular location. These enzymes are not man made though; they were originally used as a defense mechanism against invading viruses. Bacteria needed a defense against bacteriophages and viruses, so restriction enzymes were developed to chop invading DNA in pieces which could be destroyed in the cytoplasm. “The endonucleases are termed ‘restriction enzymes’ because they restrict the infection of bacteriophages (2).” The enzymes take the invader’s genetic information and cut it into pieces which cannot interact with the bacteria’s own genetic information. This is an effective way of protecting the bacteria from bacteriophages or from other sorts of invading bacteria, but the enzyme needs to differentiate between its own genetic material and that of and invading bacteria.

Restriction sites come into the genetic scene now. Recognition of the proper location to cut enzymes uses two different elements which are described by Karl Drlica, a PhD in Molecular Biology:

“This recognition process involves two elements. First there are specific nucleotide sequences [As and Ts, Cs and Gs] that act as targets for the nuclease. These are called the restriction sites. Second, there is a protective chemical signal that can be placed by the cell on all the target sequences that happen to occur in its own DNA. The signal modifies the DNA and prevents the nuclease from cutting. Invading DNA's, lacking the protective signal, would be chopped by the nuclease (1).”

The specific site where the enzyme can make its cut is the restriction site. The second form of defense against destroying a bacteria’s own DNA is the presence of a chemical signal. The chemical signal is in the form of a methyl base. This base is paired with either an adenine or cytosine base on the DNA. However, the methyl base pairing will not affect DNA replication or the reading of the genetic code (2). This signal is placed on the sequence of nucleotides which happen to occur naturally in the bacteria’s own genetic code; thus, the enzyme cannot make its cut on the DNA. These elements of defense against the restriction enzymes are needed because restriction enzymes are so effective and precise in cutting DNA. This is exactly why the discovery of restriction enzymes was an integral part of furthering the scientific advances in the bionanotechnology field.

Restriction enzymes do two things, read and cut. Restriction enzymes are like a special form of DNAase, but the main difference is that restriction enzymes cut the DNA in a desired location, not just randomly. The enzyme reads the sequence of the DNA backbones, searching for a specific series which it would then bind itself to. The recognition sequence is normally four to six nucleotides long, and they are complementary to each other. This allows the same enzyme to cut both strands of DNA (3). The enzyme then separates the DNA in a specific way, either leaving a blunt end, or sticky end. A blunt end of cut DNA is a straight cut, which means that the DNA was cut in the exact same location on both the 5’ and the 3’ sides.

5'-CpTpGpApTpCpTpGpApCpTp GpApTpGpCpGpTpApTpGpCpTpApGpT-3'

3'-GpApCpTpApGpApCpTpGpAp CpTpApCpGpCpApTpApCpGpApTpCpA-5'

Figure 1. A blunt end cut on the DNA (5)

Sticky ends are created when the restriction enzyme cuts the 5’ and the 3’ sides at different locations on the DNA. The incisions are staggered so the opposing sides of the DNA have overhangs. The sticky end cuts are the most common type of cut on anti-parallel strands of DNA (3).

5'-ApTpCpTpGpApCpT        pGpApTpGpCpGpTpApTpGpCpT-3'

3'-TpApGpApCpTpGpApCpTpApCpGp        CpApTpApCpGpA-5'

Figure 2. A sticky end cut (5)

The reason that the restriction enzymes play such a major role in genetic engineering is because of the possibilities sharing DNA. The pieces of DNA which were cut by using restriction enzymes can then be bonded with any from of DNA which was cut with the same restriction enzyme. This way DNA from different sources can be bonded together, after they have been cut by using the same restriction enzyme.

Figure 3. EcoR1 restriction enzyme (4)

Figure 2 shows how the restriction enzyme EcoR1 can cut the DNA from two different sources. The restriction enzyme is characterized by the Pacman figure. Since the two different sources of DNA had been cut by using the same restriction enzyme, these strands of DNA can then be bonded together. The newly formed genetic material is called recombinant DNA. From here the recombinant DNA can have its genetic code translated into proteins. In essence, recombinant DNA is the heart of genetic engineering, and this is only made possible by the use of restriction enzymes.

Restriction enzymes are like the oil in a car. The engine cannot run without it, and in this case the engine is genetic engineering. Scientists have been able to harness the power of this molecular machine just like a bacteria cell uses it to fend off invading virus and other malicious strands of DNA. Restriction enzymes only have two tasks to perform, and they have become so efficient in completing these tasks with a high level of accuracy, that the future could be altered by the power which they could possibly create. A possible solution to global warming could be in the form of something which can’t even be seen by the naked eye. Restriction enzymes are a molecular machine which when combined with other scientific advances, like recombinant DNA, can create endless possibilities on the micro-scale level.
Works Cited

1. “Chapter 3: Genetic Engineering.” Science Clarified. 20 February 2008 .

2. Goodsell, David S. “Restriction Enzymes.” RCSB Protein Data Bank. 20 February 2008. .

3. “Restriction Endonucleases.” The McGraw-Hill Companies, Inc. 20 February 2008 .

4. “Restriction Enzyme - Action of EcoRI.” Access Excellence at the Nation Health Museum Resource Center. 22 February 2008. <http://www.accessexcellence.org/RC/VL/GG/restriction.html>.

5. “Restriction enzyme.” Bio-Medicine.Org. 20 February 2008 .

6. United States Environmental Protection Agency. “Recent Climate Change. . .” 8 February 2008 .

sorry

Sorry guys, I can't figure out how to blow up the font. I copied it and blew it up to the biggest size but when I posted, it stayed tiny. You might have to copy it and paste it somewhere, then blow it up bigger. Sorry.
It is money though

Tyler

ATP Synthase that you can actually read

ATP Synthase

ATP
-ATP produced by adding a Phosphate to ADP
-universal energy currency

ATP Synthase
Structure
F0- handles flow of hydrogen ions. Contains three proteins. Couples ATP Synthase to the membrane
F1- made of many subunits, where synthesis takes place

How it works
Hydrogen ions pumped through a proton pump to a higher concentration.
Can only come back through ATP Synthase
This force gives ATP Synthase its energy

Formation
Three subunits that rotate in a cylindrical formation
Rotation causes change in structure, and thus in function
The F1 binding ability changes as structure changes
Still binds an ATP with every rotation

Uniqueness of ATP Synthase
Combining ADP and Phosphate does not require energy -> Binding of ADP and Phosphate to the same enzyme does
* unique because in other enzymes, reaction requires energy

Aplication
-Can use ATP synthase to power inorganic nickel
-In extremely large quantities, could use to help to power
Help in the transition from oil to hydrogen power

ATP Synthase

ATP Synthase

ATP
-ATP produced by adding a Phosphate to ADP
-universal energy currency

ATP Synthase
Structure
F0- handles flow of hydrogen ions. Contains three proteins. Couples ATP Synthase to the membrane
F1- made of many subunits, where synthesis takes place

How it works
Hydrogen ions pumped through a proton pump to a higher concentration.
Can only come back through ATP Synthase
This force gives ATP Synthase its energy

Formation
Three subunits that rotate in a cylindrical formation
Rotation causes change in structure, and thus in function
The F1 binding ability changes as structure changes
Still binds an ATP with every rotation

Uniqueness of ATP Synthase
Combining ADP and Phosphate does not require energy -> Binding of ADP and Phosphate to the same enzyme does
* unique because in other enzymes, reaction requires energy

Aplication
-Can use ATP synthase to power inorganic nickel
-In extremely large quantities, could use to help to power
Help in the transition from oil to hydrogen power

Chapter 4a Summary

The first section of Chapter 4 addresses the forces and stability of molecules that we do not necessarily notice on a macroscale level. The major difference is simply the amount of energy involved. For bionanotechnology to work, it must comply with the laws of the forces of attraction between molecules and atoms. This information, most of which has already been researched thoroughly by biologists, chemists, and physicists, is essential to properly designing molecular machines for functionality. One factor that changes the way molecular machines work is their environment; water is the necessary solution that almost all molecular machines absolutely must exist in to function. In addition, there is a hierarchical structure to the strategies of designing and assembling molecular machines: 1) sequential covalent syntheses - where each atom is placed individually until the whole is completed; 2) covalent polymerization; 3) self-organizing synthesis; 4) self assembly. This hierarchy also aligns itself in an obvious continuum of completely dependent upon human direction to independent construction, which would become very valuable in mass producing molecular machines.

BioBotz

Before that presentation I did not realize how far the group had come in their venture. I was amazed that within a year they had already been awarded multiple grants and have a chance at a huge $250,000 McArthur grant. Obviously this kind of achievment requires much passion, work, and dedication. They are very organized, hiring artists and web designers when needed and they seem to be moving quickly with their ideas. I especially like how they are targeting multiple areas of kids' lives such as video games, toys, and storybooks. I feel that kids will find the cartoonish characters fun and interesting, and they can learn about molecular machines without feeling like they are "learning" but simply by engaged exposure to them. I can definitely appreciate what this group is doing and I hope they can continue to move forward with this project.

BioBotz Presentation

Today's presentation was very informative. I'm trying to decide if its sad that I was interested in playing the video game they were discussing...Regardless of that I really loved how simple they made a concept like molecular machines seem. The plan to make all this information available to kids at a young age can only do good for their futures. I know if I was exposed to this sort of information at a younger age I would have already know as much or more than I do now because of my lack of exposure to nano-technology until I reached Wake Forest. An idea to get kids thinking about important research areas before they are even in high school can only help to improve the results our nation, and the world will notice in an field such as nano-technology. This will help to increase general public knowledge and hopefully create more future nano-tech researchers. The BioBotz are going to do so much good for general public knowledge and for the furthering of research and development of molecular machines.

BioBotz Presentation

Today's presentation on BioBotz was interesting and motivating. Their presentation showed me how much time and effort they have put into their business plan. I could tell that they really knew their material when it came time to explain exactly what the Molecular Machine was, and what it does. Their ability to transform such a simple idea of getting children interested in molecular biology, into a potentially large business adventure is motivating. It also demonstrates that what we learn in class can actually be used for something more than tests and papers. They were very open with their ideas, and as a class we had some sort of input on what their website design, which made me realize that I could be like them next year.