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Background:

Environmental toxins, including metals and organic molecules, have diverse effects on organisms. In order to compensate for the toxic effects of a given toxin, an organism may increase production (up-regulation) of enzymes that may metabolize or modify the toxin in order to neutralize it, membrane transport proteins that may pump the toxin out of the cell, sequestering proteins that bind and effectively remove the toxin, or increases in levels of proteins damaged by the toxin in order to compensate for loss of protein function.

Increasing levels of a given protein in a cell can be done by several mechanisms:

1. Increasing transcription of the gene coding for a protein.
Changes in transcription will result in increases of the messenger RNA for that protein, which can be detected by techniques such as RT-PCR.


2. Increasing stability of a given mRNA so it can be repeatedly read by the ribosomes, resulting in more protein produced.
This type of regulation would not be detected at the level of mRNAs by RT-PCR, since more mRNAs are not being produced, but levels of the protein still increase.

3. Increasing the level of translation (protein synthesis) of a given mRNA, resulting in more protein being produced.
Once again, this type of regulation would not be detected by RT-PCR, as no changes in the level of the mRNA occur, but ribosomes are reading them more efficiently. Numerous proteins bind to mRNAs that either promote or inhibit translation by the ribosomes.

Therefore, investigating mRNA levels detects only regulation at the level of transcription and can miss other means of regulation that can alter levels of a given protein. Using methods that detect the proteins directly can provide insights into a cell or organism/s response to toxins that are missed by RNA-only methods.

2-Dimensional Gel Electrophoresis:
One of the most powerful techniques to analyze total protein expression profiles in cell or tissue samples in 2-dimensional gel electrophoresis. This method separates proteins initially on a gel with an internal pH gradient (isoelectric focusing or IEF) where proteins stop migrating at the region of the gel with a pH equal to that of the protein isoelectic point. This effectively separates proteins by their intrinsic electrical charge. The IEF gel is then layered on top of a second, SDS-PAGE slab gel, which separates proteins by their molecular weight. The result is a 2-D plot with hundreds of spots, each spot representing a protein of a given molecular weight and isoelectric point. The method is so sensitive that single amino acid substitutions can be detected. Because of limits on the sensitivity of staining and detection of proteins, 2-D gels cannot detect very low abundance proteins.

Technological improvements have made the use and analysis of 2-D gels much easier and more powerful. The IEF gels are now run on IPG strips, pre-made small strips that offer both convenience and higher consistency than the previous IEF tube gels that were subject to stretching and damage during handling, and inconsistencies during preparation. Many manufacturers offer pre-made, consistent SDS-PAGE gels ready for use. Software packages allow quantification of each spot and digital overlaying and comparisons between two or more gels and, therefore, allow detection of new or missing proteins and changes in the level of a specific protein between two or more gels. These new technologies make 2-D gel analysis a powerful tool for determining the effects of treatments or experimental conditions on the expression of proteins.

A major challenge in 2-D gels is to properly dissolve out all the proteins in a sample in order to get a representative sample of proteins and sufficient proteins to get numerous spots on a 2-D gel. We are currently developing and optimizing methods for sample extraction and solubilization.

This photo shows a 2-D gel of control proteins from E. coli. Each spot represents a protein of a given isoelectric point (charge) and molecular weight.

Potential Projects:

Our current projects are described below, but use of 2-D gels can analyze a wide range of effects of toxins or environmental conditions on cells, animals or plants. New ideas for projects are encouraged!

1. Effects of exposure of Snow Creek water on developing frog embryos:

The Monsanto (now Solutia) plant in Anniston, AL produced polychlorinated biphenyls (PCBs) from the 1920s to the 1970s, resulting in extensive contamination of local soils and watersheds with PCBs. The plant is an EPA Superfund site, and the surrounding region is regarded as one of the most heavily contaminated in the nation. Snow Creek is a small stream that originates near the Solutia plant and eventually flows through Anniston and into Choccolocco Creek, which empties into Logan Martin Lake some 25 miles downstream. Snow Creek is heavily contaminated both with PCBs and with mercury from nearby foundries (the sources of the mercury are downstream from our collection site). We raise Xenopus laevis frog embryos in control solutions or Snow Creek water, and observe the effects over time. Raising embryos from the late blastula stage through 96 hours, where most organs are developed, is the basis for the widely used FETAX (Frog Embryo Teratogenesis Assay-Xenopus) assay. Our data shows a subtle but statistically significant inhibition of growth of the embryos raised in Snow Creek water. This could be due to toxins, different minerals in the water, or other effects. We are currently attempting to compare protein profiles from control and experimental embryos by 2-D gel electrophoresis to determine if there are consistent and reproducible changes in levels of specific proteins.


2. Effects of lead exposure on Kudzu tissues.

Plants cannot move away from environmental stresses and, therefore, have developed a number of biochemical mechanisms to deal with the them. Exposure to heavy metals, such as lead, have broad toxic effects on plants, But plants exposed to metals produce high amounts of small organic molecules called phytochelatins to remove the metals from circulation inside the cell. The project that soon will start involved extracting proteins from control and lead-exposed Kudzu plants. Although most plants exposed to heavy metals have the highest concentrations in their roots, the roots of Kudzu are woody and difficult to extract for 2-D gel analysis. We may use stems or even leaves for this investigation.

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