Examined Life: October 2005

Friday, October 28, 2005

Plasmodium versus the Sickle Cell Mutant

Malaria is a disease caused by infection with one of the few species of the parasite, Plasmodium, often Plasmodium falciparum (the others being vivax, ovale, and malariae ). Plasmodium is transmited by the bite of a female blood-sucking mosquito, each species of plasmodium has its own species of mosquito that acts as a vector. The parasite then goes thru a series of physical changes and stages as its life cycle progresses, residing in Red Blood Cells in one stage. They reproduce inside a person's blood stream and body, making a person host to a thriving population of them. This causes, as might be imagined, all sorts of problems, which is what the disease malaria is. Eventually some of them will leave the human body by being picked up by other blood-sucking mosquitos, thru which they spread to other hosts.
Sickle Cell Anemia is a disease of the blood, wherein, because of a point mutation, the hemogloblin molecules in the Red Blood Cells (RBCs) aggregate; they tend to stick to each other, forming long chains. This deforms the RBCs, making them shaped like little sickles. This effects the oxygen carrying capacity of RBCs negatively, and also makes it difficult for them to move thru the smallest of the bodies capillaries; they tend to jam up inside them and prevent bloodflow.
The deformed RBCs are not proper homes for the malaria parasite, it cannot live in them. Without being able to do this, the parasitic infection is greatly reduced and the disease can be prevented from developing in an infected person all together. ( sickle-cell haemoglobin )

Physical Mechanism of Resistance
In 1978 Martin Friedman (1) of Rockefeller University, was investigating the mechanism of this known resistance. He was able to do this because of new advances in the ability to culture RBCs infected with falciparum. sdgfsdfs He grew the parasite in these new cell cultures, with RBCs from normal, heterzygous for sickle-cell, and homozygous for sickle cell, individuals, both under normal Oxygen concentrations and low oxygen concentrations, with sickle celling being induced by low concentrations. He found that the parasite was not effected by cells that hadn't gone sickle. Under the low oxygen concentrations, the parasites in normal cells were relatively normal, the parasites in heterozygous sickle-cell cells were inhibited, and the parasites in homozygous sickle-cells were destroyed. The mechanism of destruction in homozygous cells was found to be that the chains of deformed hemoglobin would function like needles, actually rupturing and puncturing the bodies of the parasite. In heterozygous sickle-cells however, a different mechanism was at work. Freidman couldn't identify the mechanism, however he noted that these parasites were very similar to parasites grown under 'poor' conditions, indicating that there was something that was inhibiting their metabolism at work. A 1979 (2) study by Friedman also examined the physical destruction of the parasite, finding that disintegration of the membranes and cytoplasm of the parasite following their puncturing. In heterozygous cells Freidman observed vacuolization of the parasites, again indicating that a metabolic mechanism was inhibiting their growth.
Kodjo Ayi et al (3) completed a study that found that RBCs that were infected by Plasmodium and that had the sickle cell trait, or indeed any of a number of blood hemoglobin diseases such as thalessima, "homozygous hemoglobin C (Hb-C), and glucose-6-phosphate dehydrogenase (G6PD) deficiency", were preferentially phagocytosised by monocytes, particularly in the signet-ring stage of the parasite.
Shear et al (4) created transenic mice that expressed fetal hemoglobin in their related study of the disease, finding that the parasite can't properly digest fetal hemoglobin, and reasoning that this is also a protective mechanism in sickle-cell anemia.
Becker et al (5), in a recent study, found that the parasites are "highly susceptible to alterations in the redox equilibrium", that is, on the balances of reactive oxygen species, charged ions and compounds of oxygen. They note that the immune response to infestation with Plasmodium is to destroy the RBCS that are infected and also to create nitric oxide and oxygen radicals. The parasite, for its own part, metabolizes haemoglobin into free haeme and hydrogen peroxide, which damages the cell and might also set up a finely-tunned redox equilibrium, which permits the parasite to function normally. The production of radicals by the immune system along with the inability to properly metabolise the chained, malformed sickle-cell haemoglobin, throws off that balance.
Williams et al (6) concluded that sickle-cell affords protection also by enhancing the innate and acquired immunity to the parasite itself. They reasoned that if protection from malaria varies with age in sickle-cell individuals, then that might indicate that there is an immune response at work. Their study found that protection in sickle-cell individuals increased throughout the first ten years of life, and returned to a 'baseline' after wards, and noted that this is 'most likely explained' by accelerated immune acquisition. Citing Ayi's study above, they reasoned that the increased phagocytosis of RBCs containing the parasite, involving the marking of the infected RBC's membrane, is responsible for this accelerated response. They also suggested that, alternatively, since heterozygotes for sickle cell anemia have a greater number of different types of falciparum strains when they are infected, and a longer exposure to these different strains (tho they still have a reduction in symptoms), that this permits the innate immune system to 'familiarize' itself with the different types of parasites and thus be better able to deal with the repeating cycles of parasite proliferation.

Digestion of normal haemoglobin
The malarial parasite, as it resides in the RBC, metabolizes and digests haemoglobin into consituents, one of which is Ferriprotoporphyrin IX (aka FP). (7) This product is detoxified via biomineralization by to a compound called hemozoin. Chloroquine, an antimalarial agent, prevents this detoxification, FP accumulates in the membranes and kills the parasite. The chloroquine does this by comepetively interfereing with glutathione.

Quinine, however, is the historically traditional drug used to treat malaria, comming from the bark of the chinchona tree. In the Imperial Age, Great Britain had outposts, forts, and colonies all over the world, including regions infested with malarial mosquitos. The British peoples, however, did not have any historical exposure to malaria, and thus any time the sickle-cell anemia popped up in their population, it represented a great loss in fitness for the individual and was subsequently suppressed and wiped out via natural selection. The British, therefore, had to develop a method of protecting their citizens and soldiers from the disease that they were being exposed to. Initially quinine was given in pill form, however the drug is hideously distasteful. Thus it was mixed with water, to dilute it and make it more palapable. This is called tonic water. Unforntunately, it too is far too distasteful to easily encourage people to drink it in the amounts needed to prevent malarial infection. Finally tonic water was mixed with gin, and thus the drug was delivered to the patients effectively. Indeed, the patients would deliver themselves to the drug.

More on malaria and Sickle-Cell anemia

If one looks at a map of the world and compares the incidences of malaria infection and the incidence of sickle cell anemia, one finds that they overlap. This is because the pressence of high transmission of malaria creates a selection pressure upon humans where resistance to malaria is favoured. Sickle Cell anemia comes about by a single point mutation, a change of a single nucleotide in a gene that codes for part of human hemoglobin. This mutation of the gene is an allele, an allele being merely any alternate form of a gene. A person can inherite this mutant allele. If they inherite one copy from their father, and one copy from their mother, then they have two copies of the mutant allele, and will have the horrendous disease known as sickle cell anemia, their body cannot produce normal hemoglobin, their RBCs sickle, and they die rather young, and painfully at that.
If they have only one copy of the mutant allele from one parent, and a normal copy of the gene from the other parent, then they are producing some hemoglobin that is normal, and some hemoglobin that is abnormal. Their RBCs will show a variation, from fully normal to fully sickled and everything in between, but they will tend to not die young from sickle cell anemia.
They will also be resistant to malaria, because their RBCs are inhospitable to the malaria parasite. This means that while others around them are dying from malaria, or, at least not producing all that many children because of having to deal with malaria, they are producing children, and, indeed, those children will have a chance to also inherit that parent's copy of the sickle cell gene, and thus their fitness will be greater than that of those around them. This means that sickle cell anemia, normally a destructive and harmful disease that would decrease an individual's fitness, is actually of great benefit in an environment where malaria is present.

The Plasmodium parasite goes thru an incredible array of body-forms during its development, not unlike how a frow hatches from an eggs as a tadpole and then later matures into an adult frog, only with more forms. Sporozoites are released from the salivary glands of the host mosquito. They enter the bloodstream and work their way into the cells of the liver. Therein they replicate as merozoites. They break out of the liver cell, bursting it, and it is now that the merozoites enter the RBCs. Once there, each merozoite divides and reamin together as a form called the Schizont. The parasite stays within the RBCs for a set period of time, different lengths for different species, anywhere from two days to three days. While invested within the RBCs, they can appear in a typcical 'signet ring' shape. After that, the schizonts rupture and the RBC itself is destroyed, wherein the merozoites move on to invade other RBCs. This rupturing causes the clinical symptoms of malaria itself, the "bone breaking" chills/fevers. This process also explains why the symptoms occur in cyclic episodes with malaria, the merozoites are destroying large batches of RBCs every few days, all at once. Mature forms of the parasite are also called trophozoites.
The parasite can also enter a different cycle, a sexually reproductive cycle, once entering the RBCs. Instead of dividing into schizonts, the merozoites can form into gametocytes, which are taken up by blood-sucking mosquitos. Once in the guy of the mosquito, each male gametocyte divides into 8 microgametes, which can fertilize the undivided female macrogamete. The fertilized form is called the ookinete, the moving egg. This egg burrows thru the guy wall and encysts on the outside of the gut wall (iow still inside the mosquito itself). The cyst eventually ruptures, spilling sporozoites into the mosquito body, which migrate and end up residing in the salivary glands, to begin the cycle renewed.
This intricate and complex cycle becomes even more complex when one considers that different species of Plasmodium are transmitted by different species of Anopheles (the genus of the mosquito vector for human malaria).

a few of the relevant stages

Cited Articles:

Saturday, October 01, 2005

Purification of the product of the 2nd PCR reaction, Digestion set up

A small sample of the 2nd PCR reaction will be run on gels, to ensure that the product was acheived. The rest of the PCR will be purified for later steps.

PCR Reaction Procedure

Pipet a small 1 microliter dot of DNA loading buffer onto a peice of parafilm
Remove 10 microliters from the PCR tube and mix with that dot via pipet
Pick up the mixed smample and load it into on fo teh wells on teh gel, using a gel with small wells. A DNA ladder is loaded into the first lane
Run the gel at 120V for 1 hour.
Stain in ethidium bromide for apporximately 15 minutes and visualize.

As the gel runs, perform the purification.

Purification of 2nd PCR product

Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. Since 40 microliters of sample used, 200 microliters of buffer is used.
Place a QIAquick spin column in a provided 2ml collection tube
Apply the sample to the QIAquick colum and centrifuge for 1 minute to bind the DNA
Discard the flow-through. Place the QIAquick column back into the same tube.
Add 0.75 ml Buffer PA to teh QIAquick column and centrifuge for 1 minute, to was the sample.
Discard flow thru and place the QIAquick column back into the same tube and centrifuge for an additional minute.
Place the QIAquick column in a clean 1.5ml microcentrifuge tube
To elute the DNA into the microcentrifuge tube, add 30 microliters of water to teh center of the QIAquick membrane, allow it to sit for 1 minute to increase the DNA concentration, then centrifuge for 1 minute.

Now the product is run thru digestion.

Digestion

Add 4 microliters of 10x Buffer#2 to the eluted DNA
Add 4 microliters of BSA to the tube
Add 2 microliters of the premade enyxme mix to the tube (composed of HindIII/XhoI)
Mix the contents ths far by setting the pipet to 25 microliters and gently pipetting up and down 2-3 times

The primers have the sites for the restriction enxyme, and will cut the sample there. Later, it will be mixed with similarly restricted bacterial plasmids. Because both the plasmid and the sample DNA has been cut in the same with, they will be able to 'stick' to one antoher, and thus the mutant DNA will recombine into the bacterial plasmid, thus priducing a recombinant, transgenic, mutant genome.

DNA extraction from Gel Slices and the 2nd PCR Reaction

The DNA bands in the gel will be extracted using a QIAquick gel extraction kit, and then a 2nd PCR reaction will be done using the purified DNA as teh template.

QIAquick Gel Extraction Protocol

Weight the gel slice in a tube, making note to subtract the weight of the tube itself.
Add 3 volumes of Buffer QG to 1 volume of gel. For this experiment it will be 630 microliters and 480 microliters for the A and B gels respectively.
Incubate at 50 degrees Celsiu for 15 minutes.
After the gel discolces completely, (noting that the mixture is a yellow colour), add 1 gel volume of isopropanol to each sample and mix.
Place the QIAquick spin colum in a 2ml collection tube and put sample in.
Centrifuge for 1 minute to bind the DNA
Discard flow-through and place QIAquick column back in teh same collection tube.
Add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 minute.
Discard the flow-through and centrifuge thet QIAquick column for an additional minute, at 13,000 rpm.
Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
Elute the DNA by adding 40 microliters of elution buffer to the center of the QIAquick column membrane. allow it to stand for 1 minute, then centrifuge for 1 minute.

2nd PCR Reaction Protocol

Into a single eppendorf tube, add via pipet:

40.5 microliters of H2O
1 microliter of Primer 1 ((the forward start primer)
1 microliter of Primer 2 (the reverse primer with teh stop codon)
1 microliter of dNTPs
1 microliter of the 10X PCR Buffer
1 microliter of Vent Polymerase
1 microliter of the template DNA from PCR A
1 microliter of the template DNA from PCR B
Mix by pipeting 40 microliter volumes 2-3 times.

The toal volume should now be 51 microliters. Place this mixture into the PCR machine, set at 95 degrees Celsius. Then have one cycle of 5 minutes of denaturation at 95 degrees Celsius. Then have 30 repitions of this set:

Denaturation at 95 degrees Celsius for 1 minute
Annealing at 50 degress Celsisus for 1 minute
Extension at 72 degress Celsisus for 1 minute

Followed by a single 10 minute extension at 72 degress Celsisus.

Gel Electrophoresis of PCR Products and DNA Purification

Continuing with the mutation of the VHL tumor suppressing gene experiment, the previous PCR products will ge run on agrose gels, and the PCR DNA will be purified for later use.

40 microliters of the PCR product will be run on teh gel via the following method.

Pipet 2 small dots containing 4 microliters of DNA loading buffer onto a peice of parafilm.
For PCR A, remove 40 microliters from the PCR tube by pipet and mix with the 4 microliters of DNA loading buffer by pipetting up and down a few times on the parafilm
Pick up the mixed sample and load it inot one of the large wells on the gel, using the 10-100 microliter pipette, releasing slowly
Repeat steps 2+3 for the PCR B. Leave an empty well between A and B PCR samples. There will be other samples from other groups sharing the same gel. A DNA ladder will be put into the first lane.
Gels will run at 200 Volts for 20-30 minutes.
The gel is then stained in ethidium bromide for approximately 20-30 minutes and then visualized under ultraviolet light.
The glowing band of DNA will be cut out with a scalpel, taking care to get as little of the gel and as much of the DNA as possible.
The two cut out bands will be placed into seperate eppendorf tubes, labeled, and will be used to extract the DNA itself later.

Unfortunately, upon following this procedure, it was found that no DNA from teh original PCR was present. There was no banding seen on the agarose gel, for any of the group samples, whereas the ladder did show banding. The staining was permited to continue for approximately another 20 minutes, with new concentrated ethidium bromide added to the staining solution, still the PCR bands failed to show up, stronly indicating that there simply was no PCR DNA.

Upon review, it was discovered that the orignal stage of the experiment was missing a critical component, the dNTPs, from which the PCR DNA is built out of.

The orignal stages were repeated with the correct procedure. After repeating the steps above, the bands did appear, and those bands were cut out and set aside in eppendorf tubes for later stages.

While the above gel was running for the second time, the remainder of the DNA (approx 40microliters) was purified using columns and buffers from the QIAquick PCR purification kit, using the following procedure.

Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. Since 40 microliters of sample are left, 200 microliters of buffer is used.
Place a QIAquick spin column in a provided 2ml collection tube
Apply the sample to the QIAquick colum and centrifuge fro 1 minute to bind the DNA
Discard the flow-through. Place teh QIAquick column back into teh same tube.
Add 0.75 ml Buffer PA to teh QIAquick column and centrifuge for 1 minute, to was the sample.
Discard flow thru and place the QIAquick column back into the same tube and centrifuge for an additional minute.
Place the QIAquick column in a clean 1.5ml microcentrifuge tube
To elute the DNA into the microcentrifuge tube, add 30 microliters of water to teh center of the QIAquick membrane, allow it to sit for o1 minute to increase the DNA concentration, then centrifuge for 1 minute.

Now a restriction enzyme digest of this purified DNA will be set up, using the following protocol:

Add 4 microliters of 10x Buffer#2 to the eluted DNA
Add 4 microliters of BSA to the tube
Add 2 microliters of the premade enyxme mix to teh tube (composed of HindIII/XhoI)
Mix the contents ths far by setting the pipet to 25 microliters and gently pipetting up and down 2-3 times

What is now had is a tube containing a total volume of 40 microliters (the 309 microliter water+DNA sample, and the combined 10 microliters above). It is important to not hold the base of the microcentrifuge tube and thus warm it, because the restriction enzyme are refrigerated and are very heat sensitive.

This tube is now incubated at 37 degrees Celsius overnight, stored, and then used in later steps.

By this time the gels have run and have been stained and are ready for viewing (with UV protective face shields) via UV illumination.

The ethidium bromide is UV reactive and also binds with DNA, thus when illuminated with UV light, the locations of the DNA bands, which have moved during electrophoresis, show up as glowing bands, similar to the gel phot shown below, however in this experiment there is only a single band (excluding the bands in the ladder DNA row), because only one fragment has been prepated (per row, the PCR row A, and PCR row B). It is these bands that are now cut out and placed into eppendorf tubes for later procedures.