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Effect of environmental toxins on GATC methylation in E. coli May 3, 2011

Posted by ljsteele in Biology, Chemistry, Ecology, Environment/Conservation, Evolution, Genetics, Health, Marian University curriculum, Physiology.
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With the end of the semester drawing near it is becoming that time again when the results are piling in from research you have been working on all semester. As we speak, the final data collection and analysis is taking place in biochemistry, a team of student researchers are exploring of environmental toxins of DNA methylation  in the bacterium E. coli. 

The Bacterial Genome

Bacteria exist throughout the world and can survive in almost any climate . Bacteria are unicellular and can consist in a wide range of environments such as a pond all the way to soil.  One unique attribute of the bacterial genome is that it contains adenine methylation , opposed to mammalian organisms which contain cytosine methylation at GpC islands.  Adenine methylation is when a methyl group becomes attached to the adenine nucleotide on the DNA. When a methyl group is donated from SAM to form a covalent attachment, it is made on the adenine which can cause steric hindrance of transcription factors and differential effects of DNA binding proteins, which can contribute to a change in gene expression.  In previous studies  it has been shown when E. coli is exposed to different carbon sources (ie glycerol or glucose).  Some areas of the genome become demethylated.  In the bacteria E. coli almost every adenine (A) in the GATC sequence is methylated.  To block the methyation at the GATC sequence, a protein must be present to inhibit the DAM methyltransferase from depositing a methyl group on the adenine.

What does Methylation do?

Adenine methylation has many roles in bacteria. Methylation can effect gene expression, cell cycle, virulence, and how proteins interact with the DNA. For the research we are performing, we are concerned with what effect the environment has on changing adenine methylation on the GATC repeats. There are about 20,000 GATC repeats in the E. coli genome and under normal log growth conditions almost every single repeat is methylated. It has been found that when bacterial cells are in a log growth phase there are 6-10 sites which are not methylated. These nonmethylated sites lie up and down stream of promoters of different genes. The lack of methylation may allow DNA binding proteins to modulate their function to allow a functional change in gene expression.

Pollutants and the Genome

In the study we are performing we wanted to see how three classes of chemicals pollutants commonly found in the Midwest affect adenine methylation at the GATC site. We choose three pollutants to represent chemicals that fit into the families of common water pollutants, which are heavy metals, chlorinated compounds and nitrogen rich compounds.Gel Electrophoresis

The above families of compounds will be compared to samples collected from different areas around the campus of Marian University, Indianapolis, IN. Supplements will be added to all the samples to generate a rich liquid media that will facilitate bacterial growth.  With 6 different test groups and 2 controls we are going to seek to determine if any of our known compounds or a compound present in our environmental sample has an effect on the methylation.  The determination of methylation can be done by using restriction enzyme digest with endonuclease selecting specifically for the nonmethylated site.  The enzyme we have chosen was MBO and AVI.  When all the genomic DNA from the bacteria is extracted and digested, then it will be ran on a gel to be imaged to determine if the bands of digested DNA differ depending on the chemicals present during growth.  This is a time efficient way to examine if any changes in methylation levels have occurred.

What Does It All Mean?

For conclusion, the relevance of this study includes a few things.  This study will provide evidence to show if environmental toxins have an effect on bacterial DAM methylation. One role bacteria play in an ecosystem is influencing the flow of nutrients which support plant and algae growth. The results of our proposed study may display that toxins have an effect on methylation patterns which could lead to an increase the mutation rate of the bacteria genome itself.   Destructive mutations may decrease bacterial populations leading to a disruption in the ecosystems nutrient flow, hence disruptions in plant and algae growth with effect additional aquatic and terrestrial organisms.

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Cramming: A Student’s Best Friend? March 4, 2011

Posted by ljsteele in Behavior, Biology, Chemistry, Health, Medicine, Science & Culture, Uncategorized.
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The night flies by…

As a senior undergraduate student, slowly over the past four years I have realized the importance of cramming before a test. Simply put, by this stage in my academic career, it has become routine to stay up all night before a test to study.  In classes where there are multiple choice tests, it appears to be easier to stay up all night cramming, as is the belief that if you at least can recognize the question, ruling out the different choices for the answer becomes quite simple.  It has been shown that over a third of students cram the night before a test.

Equal Justice?

However, although many students utilize the practice of cramming, whether or not it helps students is up for debate. There are different levels of cramming, and each appear to cause different results when it comes to grades and GPA.  The issue that is starting to be seen is that although cramming may help in terms of short term memory, the retention of that information weeks after the course ends seems to be up in the air.  Of course, when cramming is being utilized, it only makes sense that the information storage would be contained in the frontal lobe of the brain, while long term memory, which would be associated with studying that has taken place over numerous days or weeks, would be stored over multiple parts of the brain.

Green highlighted area represents the frontal lobe of the brain

Many different universities have brought to light the health implications that one may bring upon him or herself when cramming. But, it is also shown that certain periods of acute stress are positive for the human body, which cramming would appear to fall under the category of acute stress. During acute stress, the body increases its fight or flight response (epinephrine and norepinephrine), shuts down digestion, reproductive systems, and boosts metabolism. Vasoconstriction and vasodilation also take place, therefore pumping blood into certain areas of the body and brain that during a normal day’s activities may not get stimulated very often.  Especially during the fight or flight response, one becomes more attentive, which would seem to help with say, studying for a huge test.

Are there more effects than just retaining information?

Although cramming may not be ideal for certain people, research needs to continue in terms of stress and cramming, and even learning styles.  Certain people are exposed to more stress than others, so possibly stress levels are compromised, leading to a decreased ability to study and cram the night before a test.  Students continue to cram because results are obtained on tests and finals.  Quite possibly cramming could do more than just get a student a good grade on a test-it could also help to train the body for different stress activities that otherwise may not be achieved.


Stress and the GI Tract December 17, 2010

Posted by ljsteele in Behavior, Biology, Chemistry, Ecology, Environment/Conservation, Health, Neuroscience.
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The mechanics of stress and the gut.

Stress is shown to have a huge effect on the body, whether or not it is experienced as an acute or chronic stress.  A major topic of interest is what effects stress has on the gastrointestinal tract in organisms.  According to a multi-part scientific paper entitled “Stress and the Gastrointestinal Tract”, there are many different stressors that can be examined within a variety of organisms.  Examples of the stressors explored include food deprivation, fearful sounds, weather changes, and water avoidance ( an acute stressors explored in lab organisms such as mice, rats, and guinea pigs).  It has also been shown that acute stressors in humans, such as pain exposure, anger, fear, and intense exercise can cause gastrointestinal shut down.

From the stressors listed above, research has explored how stress influences gastro muscles to slow contraction,  thus inhibiting the processing of food.  An interesting reaction to this slowing of peristaltic movement is the fact that many organisms lose control of their colon, showing defecation in response to certain stimuli such as fear and water avoidance.  Corticotropin releasing hormone, also known as CRH  (or CRF, as identified in the aforementioned paper), is released from the hypothalamus, and blocks the effects of the vagas nerve, while also traveling through the solar plexus, and attaching to receptors in the stomach.

Stomach ulcer

Once bound, this hormone has been shown to inhibit gastro movement, and thus preventing emptying of the stomach. The difference between the stomach and the colon is that the stomach requires contraction of the muscles to push food through, whereas the colon requires contraction to keep bowel movements inside the body.  With the effect of CRH binding to the receptors, relaxation in gastrointestinal muscles occur, which is why the release of the colon sphincter results.  However, the results explored here were in response  to short term stressors.  The effects of long-term stressors have yet to be studied.

Stress and Ulcers

What does this research mean to you?  Well, the results we glean from research like this offer powerful implications for human medicine and today’s society.  Many people not only experience acute stress, but chronic stress as well.   Short term affects of acute stress include accelerated of heartbeat and an increase in metabolism, but it is only natural to ponder the long term effects of chronic stress.    We can extrapolate from the results of  acute stress that it would make sense that we, as humans, would not want these effects to be long lasting.  Major problems would arise with the decrease of gastro movement.  Problems manifest from a build up of bile and stomach acids in the stomach.  Since gut motility is decreased when stressed, less movement would mean that more bile, which is highly acidic, would sit in the stomach longer and could lead to stomach or intestinal ulcers.

Sympathetic Nervous System

Chronic stress can also lead to a decrease in the immune system of the organism as well as a decrease in the second messenger systems within the body.  An example of this effect on a second messenger system is the attachment of CRH to CRH-receptors in the solar plexus.  The binding of these receptors causes the effect of the decrease in gastro movement.

How much stress is too much stress?

Lastly, with chronic stress and chronic stimulation of the CRH/CRF system, we might see a scenario in that the more that these receptors are activated, the more desensatized they can become. This could cause problems for people and their response to stress.  If the are “desensitized” this may mean that these people have a problem when trying to   properly responding to an acute stressors when needed.