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HHMI Undergraduate Summer Research Program
Izoduwa Abbe, LSU (David Foltz, Biological Sciences) Although sea stars of the family Asterinidae have been the subject of much molecular phylogenetic research in the past decade, several basic taxonomic and phylogenetic questions remain unresolved. In particular, it is unclear which genera belong within the family, and the monophyly of the Asterinidae has never been demonstrated on either morphological or molecular grounds. Here, previously-published mitochondrial DNA sequence for two rDNA-coding genes, plus some flanking tRNA sequence, was used to design PCR primers for amplification and sequencing of around 1.2 kilobase of DNA for 21 sea star species, and comparable data were obtained from GenBank for an additional 21 species. The species selected for phylogenetic analysis included representatives of 18 genera believed to be members of the family Asterinidae, plus a few additional species and an appropriate outgroup species. The neighbor-joining algorithm in the computer program MEGA v. 4 was used to reconstruct a molecular phylogeny for these sequences and test for monophyly via bootstrap analysis. Several monophyletic groups were recovered with >80% bootstrap support, but some of the recovered groups did not correspond to traditional taxonomic categories. Nina Allemond, LSU (Marcia Newcomer, Biological Sciences) 8R-Lipoxygenase is an enzyme found in the sea whip coral Plexaura homomalla which catalyzes the dioxygenation of polyunsaturated fatty acids. The enzyme has a C2-like membrane binding domain that requires calcium for activity. The diminished capacity to bind calcium thus leads to a lower membrane binding ability, but it is also believed that this same calcium plays a role in enzymatic activity. By mutating amino acid sequences that are involved in calcium binding (W77, D80, T18, and D19) we set out to determine the effect that calcium has on the enzymes activity in a membrane-less assay. We made these mutations in the vector Pet-3a with whole plasmid polymerase chain reaction (PCR) and inserted silent mutations into the PCR primers so that we could later determine mutant 8R-LOX’s presence in the vector. After the mutagenesis was successfully achieved, we transformed the plasmids into BL21 E.coli, which we then used to express the proteins. W77A over expressed well, while D80A and T18S/D19K did not over express well. Due to time constraints we were unable to purify the protein, and thus determine of these mutations will affect enzyme activity. We also looked at several other 8R-LOX mutants that had been previously made in the Bartlett lab. We measured the stability of these mutants (I433A, loop, S377F, and I433W) in the presence of varying levels of Guanidinium with a spectrofluorometer. It was determined that proteins with inflections at lower levels of guanidinium (S377F), were less stable than the mutant enzymes that had an inflection at higher molar concentrations (I433A). Nimalka Bandara, Wofford College (James E. Miller, LSU School of Veterinary Medicine) Internal parasites in small ruminants are a continuing problem for farmers worldwide. The most damaging abomasal parasite is the Haemonchus contortus, or the barberpole worm. With the increasing occurrence of these worms becoming resistant to almost all the available anthelmintics, non-chemical control is being investigated. Condensed tannin (CT) containing plants are one of those. The experiment was to verify that the CT in sericea lespedeza does have an effect on fecal egg count and egg hatch and/or development of larvae in the feces, as reported by previous studies. The results indicated that there was no significant effect on either. Brittany Bond, LSU (Doug Gilman, Chemistry) Capillary Electrophoresis (CE) has become one of the most attractive separation methods for oligonucleotides based on advantages over conventional methods [1-2]. In this work, we are using CE with UV absorbance detection as a separation and detection method to study native and caged oligonucleotides. Modified (caged and flashed) and unmodified (native) oligonucleotides were separated by free-solution capillary electrophoresis with higher speed and efficiency than the slab gel electrophoresis and HPLC methods that have traditionally been used to analyze these molecules. Separation of these oligonucleotides is possible because the photosensitive caging compound used, 1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester (DMNPE), modifies the charge-to-friction ratio of DNA thereby altering the electrophoretic mobility [3-5]. CE was found to be a viable alternative to conventional methods of separation and analysis of caged oligonucleotides. Erin Conlin, LSU (Mark Batzer, Biological Sciences) The Basques are an ethnic group living in parts of Spain and France whose evolutionary history is ambiguous at best. Genetic tests using classical DNA markers, such as mitochondrial DNA and Y-chromosome analyses, have failed to clarify the situation. Previous studies have had mixed conclusions, but most agree that further studies should be performed on Basques using new methods in order to reach a consensus. Recent Alu element insertions may be particularly useful in this case because they can be polymorphic within the human genome, they are considered identical by descent, the ancestral state is known, they are not subject to mutation at a significant rate, and they are autosomal. We used the Allele-Specific Alu PCR method to find new Alu insertion polymorphisms that could be used to study the Basques. We have found 3 novel polymorphic Alu insertion loci, and plan to continue this study. Myra Dennis, Furman University (Evanna Gleason, Biological Sciences) The retina is composed of a complex system of neuronal layers. Amacrine cells are interneurons located in the inner nuclear and inner plexiform layers that contribute significantly to retinal function. Nitric oxide (NO) is a gaseous signaling molecule that has been found to signal changes in amacrine cell function by triggering intracellular chloride release. Though the exact mechanism of chloride release is unclear, members of the CLC family of chloride transporters might mediate internal chloride release from endosomes, organelles known to contain high concentrations of chloride. In the experiments presented here, we used immunocytochemistry to examine the expression pattern and localization of three CLC transporter proteins. Shawn Doyle, LSU (Brent Christner, Biological Sciences) The cardinal temperatures are the minimum , optimal, and maximum temperatures which permit growth. These temperatures vary widely based on the organism. Some psychrophiles are able to grow at sub-zero temperatures while some hyperthermophiles are known to have the ability to grow at temperatures exceeding 100°C. While the range of temperatures of which life is known to have the ability to grow is very wide, no known organism exists which can grow over the entire range. The normal range between the maximum and minimum cardinal temperatures for any given organism is 30° to 40°C. The cardinal temperatures however may not indicate the widest range at which a microorganism is metabolically active. Some microorganisms may have the ability to remain metabolically active well below their minimum cardinal temperature. James Haley, Earlham College (William Doerrler, Biological Sciences) The gene encoding Bacillus subtilis phosphatidyl serine synthase (BpssA) was overexpressed in the Escherichia coli double- knockout mutant BC202. BC202 harbors deletions of yqjA and yghB resulting in altered phospholipid composition, temperature sensitivity and defects in cell division. BpssA overexpression did not restore the ability of BC202 to grow at 42 °C, nor did it result in restoration of wild type cell division. Contrary to what was reported by another group, significant changes in the levels of the membrane lipids were not observed following overexpression of pssA. Christopher Kennedy, LSU (Todd Monroe, Biological Engineering) Photocleavable cage groups can be used to control the bioactivity of nucleic acid oligonucleotides, whereby hybridization and protein interactions are blocked until the complex is exposed to near-ultra violet light. The degree of biochemical inactivation and photo-restoration are highly dependent upon the number of cage groups attached to the target oligonucleotide. The current means of caging DNA oligonucleotides is a batch-type reaction, that results in products with varying degree of cage attachment. The goal of this project is to achieve a separation between native, lightly caged, and heavily caged oligonucleotides to isolate the target caged species using column chromatography. Caged oligonucleotides isolated in column chromatography can be differentiated from native DNA and flashed DNA using a molecular beacon assay or hybridization melt curve analysis. DNA species can be fractionated based on differences in their column affinity when eluted with increasing molarity of ammonium acetate buffers from sepharose columns. Caged oligonucleotides were released from the column in the 10 mM ammonium acetate buffer. Native samples were released from the column in a 1 M ammonium acetate buffer. In further experiments different concentrations of ammonium acetate buffers will be used to optimize the separation of oligonucleotides by the number of attached cage groups. Together this separation strategy and characterization methods may be used to select caged oligonucleotides with optimal degree of caging for use in biological systems. Libby Lastrapes, LSU (Grover Waldrop, Biological Sciences) Propionic acidemia, an inborn errror of metabolism, is caused by mutations in the biotin dependent enzyme, propionyl-CoA carboxylase. To study the mutations, homologous mutations were created in E. coli biotin carboxylase. Site directed mutagenesis was used to generate the following mutatnts: alanine to a threonine at position 77, an isoleucine to a theonine at position 103, and a glutamine to an arginine at position 237. The mutant proteins were overexpressed in BL21 (DE3) and purified by nickel affinity chromatography. The activity of the mutant enzymes was measured and compared to the wild type. If the kinetic analysis can be correlated to the clinical phenotype, biotin added to the diets of the patients may be beneficial in the treatment of propionic acidemia. Leah Muller, LSU (Todd Monroe, Biological Engineering) The basis of this project is to design photoactivatable caged compounds that are biochemically inert until exposed to near-ultra violet light. The most common form of nitrobenzyl-caged compounds are themselves inert, yet they can inhibit enzymes and other biochemical processes. Using ATP as an example nucleotide, we propose novel sites of alkylation that are biochemically inert until photocleaved. These forms of caged ATP would allow more accurate control and study of kinase and other enzymatic processes. The caged ATP has been synthesized and purified, and the results presented here are contributions to the characterization of the molecule. Characterization of this compound was focused on inspecting the light intensity, wavelength, time required to uncage the compound. These properties were monitored using absorbance spectrophotometry, high performance liquid chromatography, mass spectrometry and NMR. The use of this novel cage could give a more accurate value of biochemical kinetics rates of reactions, and it could also provide a basis for further research using similar structural compounds. Caging of biological molecules has the potential for use in many applications, from studying reaction kinetics to site-specific biochemical and cellular activity, to targeted therapeutics. Thus new forms of photoactivatable biomolecules that are completely inert and stable in the caged form, and readily photocleaved with near-UV light would enhance aforementioned studies. Ha Pham, College of St. Benedict & St. John University (Jayne Garno, Chemistry) Ashley Phipps, Whittier College (Doug Gilman, Chemistry) Capillary electrophoresis was used to separate and detect folic acid in small sample volumes. Folic acid is a vitamin with great biomedical importance in terms of birth defects and possible cancer screening. To perform the separations, a simple borate buffer was used as the running buffer, running the system at 25 kV. UV absorbance detection was done at 280 nm, injecting different concentrations of folic acid at 10 kV for five seconds, with a current of 14.6 μA allowing a calibration to be performed. Fluorescence detection was then attempted using NDA and NBD-F as fluorogenic labeling reagents. Using an argon ion laser at 488 nm, NDA was used to successfully derivatize the amino acid Glycine and NBD-F was able to derivatize Glutamic Acid. However, the folic acid samples were not successfully derivatized. Kelly Pipkin, Mercer University (Steven Barker, LSU School of Veterinary Medicine) Ayahuasca, a psychoactive decoction prepared from Banisteriopsis caapi and Psychotria viridis found throughout the Amazon, is used primarily as a sacrament in ceremonial healing practices by syncretic religious cults. Today, ayahuasca use in religious practices is growing, making it important to have data through human clinical trials about the safety as well as physiological and pharmacological activity of ayahuasca in adults. Ayahuasca also has potential uses for medicinal purposes and may help treat alcoholism, drug dependence, and depresson. Thus, in order to perform sound clinical studies on ayahuasca for the future, research was done to formulate a standardized and stable preparation of ayahuasca that can be administered to humans. Twelve compounds thought to be found in ayahuasca were acquired and identified through liquid chromatography methods. Methods were created for running standards of compounds on a TSQ Quantum Access LC-MS/MS and linear standard curves were generated. The curves were used to quantify compounds detected in solutions of the extracts run in the LC. Additionally, concentration degradation of the ayahuasca extracts was measured so that the best storage conditions can be used. Results demonstrated that tetrohydroharmine, harmine, harmaline, and dimethyltryptamine are the main components of the ayahuasca extracts. 5-methoxy-dimethyltryptamine, methyltetrahydro-beta-carboline, dimethyltryptamine-N-oxide, and emetine were not detectable. Bufotenine and n-methyltryptamine were detected in trace amounts. Compounds were stable at room temperature for 48 hours and took a week to show significant decrease. This project showed that with further validation, the method is suitable for application to the characterization of ayahuasca for clinical trials. Wedad Rahman, LSU (Anne Grove, Biological Sciences) The functional roles of the HMO2 homologue can be studied by identifying the amino acid residues that are directly involved in the intercalation of DNA strands wherever double-stranded breaks have occurred. If these proteins were to play a role in DNA repair, the four largest amino acid residues of the HMO2 homologue are thought to be the key players. Through site directed mutagenesis, one of the large amino acid residues, valine (Box A, site 19) was substituted with a smaller alanine residue and expressed into protein. A binding assay with linear plasmid DNA showed that the mutated protein HMO2val19ala showed slight affinity for the DNA at only the highest concentrations whereas the wild type protein was able to bind at lower concentrations. However, the mutated protein showed similar binding affinity to its wild-type counterpart in another assay with super-coiled DNA. A protection assay was also carried out to determine valine’s ability to protect the free ends of DNA from the exonuclease enzyme. Both wild-type protein containing the valine amino acid residue and the mutated protein lacking valine were able to protect the DNA from exonuclease activity. Three assay tests were carried out to determine the mutated protein’s ability to bind to distorted DNA containing 1 loop, 2 loops and an abasic site with 1 loop respectively. HMO2 homologue did not bind to DNA with one loop but did prefer DNA with 2 loops and with 1 loop and abasic site configurations. The mutated protein in all three assays did not show much difference from the wild type protein in binding affinity for DNA in each assay. Chantal San Miguel, LSU (Vince LiCata, Biological Sciences) Similar in structure and function to the Pol I family of polymerases, Human Immunodeficiency Virus reverse transcriptase (HIV-RT) accurately replicates the HIV-1 viral genome. The hand-shaped enzyme facilitates HIV-1 propagation in the human host and is consequently the target for many antiretroviral drugs. In this study, HIV-RT was expressed and purified to homogeneity. The purified protein was structurally and thermodynamically analyzed by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). The HIV-RT CD spectrum shows it to be folded following purification and prior to heating by DSC. Following unfolding and analysis by DSC, HIV-RT lacked a discrete peak, possibly indicating protein precipitation or a lack of subunit dissociation under the present buffer conditions. The eventual objective of this project is to hopefully contribute to modern antiretroviral drug development by investigating the interaction between anti-dimerization peptides and HIV-RT. Devangi Shah, Virginia Commonwealth University (Tin-Wein Yu, Biological Sciences) Holly Stoute, LSU (Bryan Carstens, Biological Sciences) Salamanders in the Plethodon vandykei species group inhabit the mesic forests in the Pacific Northwest of North America. Mesic forests can be found in both the Cascades and the northern Rocky Mountains, and these wet forests are separated by high desert habitat in the Columbia basin. One-hundred and fifty different types of organisms have populations in both the Cascades and northern Rocky Mountains, and biogeographers have searched for the factors that have generated these disjunct distributions1. Since the entire region was severely impacted by repeated Pleistocene glaciation, the essence of this debate can be distilled to two related questions: Did population divergence occur before or after the Pleistocene glacial episodes, for example concomitantly with the desertification of the Columbia basin? Are populations in the Cascades and northern Rocky Mountains currently connected with gene flow? Carolyn Valle, University of Florida (Jayne Garno, Chemistry) Iron sulfide (Fe2S3) nanoparticles were synthesized using a new biomineralization approach with Chondroitin Sulfate type A. Samples of iron sulfide nanoparticles were characterized using tapping mode atomic force microscopy (AFM) High resolution tapping mode images of Fe2S3 nanoparticles on mica were acquired successfully. The morphology of the naturally assembled Fe2S3 is affected by the surface coverage of nanoparticles and excess CSA. At high concentration, residues of CSA and individual nanoparticles were observed on the mica. At low surface coverage, elongated aggregates of a mixture of CSA and nanoparticles were detected. Xueying Wang, University of Florida (Ken Brown, Biological Sciences) In nature, mussels produce byssal thread, which are hair-like filaments that can attach to different surfaces. Evidence suggests that blue crabs (Callinectes sapidus) select for small and solitary mussels; and constant tidal waves can damage loose mussels. Thus mussels can increase their survival rate by producing byssal thread and forming clumps. The goal of this study is to look at the effect of predation stress, mussel size, and wave action on byssal thread production. During research, hooked mussels (Ischadium recurvum) of two sizes are collected and held in four environments: agitation to simulate wave action, seawater with predator (blue crab) scent, seawater with both predator + damaged prey scent, and normal seawater as control. After 36 hours, the length and diameter of each byssal thread is measured. In the experiment, small hooked mussels produced significantly more and thicker threads than large mussels, and since smaller mussels are at higher risk for predation, the result support that increased predation stress may induce byssal thread production in hooked mussels. Rachel Wojcik, Mercyhurst College (David Donze, Biological Sciences) The genome for yeast provides researchers with a great tool for studying genetic processes because the entire genome has been mapped out and is readily available. For this experiment, double knock-outs were designed in which the TFC6 gene was replaced with URA3, followed by replacement with lacZ. This will allow for the measurement of the β-galactocidase levels after point-mutations are created in the promoter region of the TFC6 gene. At present, only the URA3 gene has successfully replaced the TFC6 and a plasmid to correct for this mutation has been inserted. Future work will be done to complete this project. Deric Griffin - EXROP Duchenne muscular dystrophy is the most common form of muscular dystrophy. It is caused by a mutation in the DMD gene on the x chromosome, which leads to the absence of dystrophin. Dystrophin is a part of the dystrophin-associated glycoprotein complex and anchors the complex, which is also bound to the extracellular matrix, to the cytoskeleton of the cell. Without dystrophin, muscle cells are destroyed due to mechanical stress from muscle contractions. The disease causes progressive muscle weakness that results in the loss of the ability to walk, and eventually affects the heart and the muscles that control breathing. The disease is 100% fatal and most men who have it do not live far into their twenties. Females can be heterozygous for the disease and are known as carriers. Female carriers manifest some of the symptoms of the disease, but not to the same degree as in males (Bernes et al). While most of the skeletal muscle fibers in afflicted males are negative for dystrophin, in female carriers, due to mosaic inactivation of the X-chromosome, some of the fibers are negative and others are positive. However, as the female carrier ages the negative fibers become less abundant. Eventually, almost the entire muscle becomes populated by positive fibers (Hoffman et al). Stem cells are cells that are capable of differentiating into many different cell types. Stem cells can be defined based on how many cell types they can become. Totipotent cells can give rise to any cells in the body including cells needed for fetal development. An example of totipotent cells would be a zygote (fertilized egg cell). Totipotent cells give rise to pluripotent cells, which are more specialized and are able to give rise to fewer cell types. An example would be embryonic stem cells. There are also multipotent, and oligopotent cells, each of which is more specialized than the former and so can give rise to fewer cell types. Stem cells have been considered as a delivery system for the correct dystrophin gene. However, results so far have been poor. Most stem cells have poor engraftment and those that do engraft do not survive for very long. In order for stem cells to be used in a clinical setting they must be easily obtained, be able to efficiently undergo myogenic conversion, and be able to integrate into muscle to produce the correct phenotype (Kuroda et al). There are two types of stem cell treatment: allogenic and autologous. These treatments are applicable for diseases that occur due to recessive loss of function. Allogenic stem cell treatment takes stem cells from a normal individual and places them in a diseased individual. The advantage to this procedure is that the cells do not have to be genetically altered, but the recipient may have an immune reaction to the cells. Autologous stem cell treatment takes stem cells from the effected individual, genetically modifies them by introducing the correct form of a gene, and then places the cells back into the same individual. This treatment lowers the chance of an immune reaction, but altering the cells could lead to transformation of the donor cells and death (Kuroda et al). The mdx5cv (x-linked dystrophy 5cv mutant) mouse is an animal model for DMD that has been used for testing these treatments. This mouse does not display as severe a phenotype as in humans, but it is a good model of the diaphragm in humans. This mouse also displays spontaneously revertant fibers, which are dystrophin negative fibers that randomly display a positive signal. In our experiment we have used stem cells, specifically muscle progenitor cells, from both a female carrier (mdx5cv+/-) and a wild type mouse (mdx5cv-/-). This makes the treatment allogenic, but the cells from the female carrier are different in the fact that they have come from an environment similar to diseased muscle. This unique property of female carrier cells may allow them to engraft better than wild type cells. We have also followed serial sections of several fibers to determine if a fiber that has been deemed negative remains so throughout its entire length. This would allow us to see if dystrophin expression is uniform at the sub-sarcolemma in fibers formed by fusion of mosaic cells. So far the experiment has yielded partial, yet interesting results. While following the serial sections in the 8 week old carrier female mouse we found that negative fibers gradually became positive for dystrophin over a span of 250 µm (fig 4). This could tell us two things in particular. For one, fibers formed by fusion of mosaic cells seem to be random. Not all positive cells fuse with positive cells and not all negative cells fuse with negative cells, it’s a combination of the two. Second, since the cells work by fusing with existing fibers this may also be able to tell us about how far dystrophin is able to diffuse throughout the fiber. Knowing how far dystrophin diffuses may aid in future studies using cell based therapies. These results have only been shown in one mouse, and so the experiment must be repeated in order to obtain any concrete results. The cell engraftment experiment must also be repeated in order to determine the approximate number of fibers that have reverted and begun spontaneously producing dystrophin. If more evidence comes along to support these results then it may lead to carrier female stem cells being used more widely in future stem cell treatments. University of Florida Collaboration Marine animals are constantly exposed to oxidative stress which triggers a range of cellular activities due to the production of free radicals. These free radicals may arrest growth, cause DNA damage, disrupt homeostasis, oxidize proteins and lipids and ultimately lead to cell death. Kurz et al (2006) had proposed that the free radicals released by hydrogen peroxide (H2 O2 ) causes lysosome membranes to destabilize by oxidative damage. They also proposed that the presence of iron, which is sequestered within lysosomes, catalyzes the formation of free radicals by the Fenton reaction. The metabolic compound hydrogen sulfide (H2S) is present in many marine habitats as the result of bacterial reduction of sulfate. H2S is highly toxic because it spontaneously produces free radicals. H2S inhibits the last enzyme in oxidative phosphorylation, cytochrome c oxidase, which leads to a decline in ATP production. Like H2 O2, H2S oxidation is catalyzed by iron, and can produce free radicals. Based on the Kurz model and the potentially similar effects of H2 O2 and H2S, we predicted that H2S destabilizes lysosomes via oxidative damage. We therefore hypothesized that antioxidants and iron chelating molecules would reduce lysosomal destabilization by H2S. We tested this using erythrocytes from the marine worm Glycera dibranchiata. We treated G. dibranchiata erythrocytes to four different sulfide concentrations (0, 0.03, 0.1 and 0.3% H2S), in the presence of three different antioxidants: glutathione ethyl ester (GEE), N-Acetyl cysteine (NAC), and hypotaurine (HT). We also tested the effect of the iron chelator desferroxiamine (DFO). Exposures were carried out in four 96-well plates. The erthythrocytes were incubated in closed chambers for two hours. We used the dye Lysotracker Red (LTR) to quantify lysosomal stability and the dyes calcein green and propidium iodide quantify cell viability. We found a direct correlation between increasing H2S and H2O2 and both cell death and lysosome destabilization, but we found that the presence of antioxidants and the iron chelator DFO did not reduce lysosomal destabilization. Our exposure to 0.3% H2S for 2 h had declined the integrated intensity of LTR by 76% (p<0.0001) and the lysosomal area by 81% (p<0.0001). We also tried the same with H2O2 and found that exposure to 0.01-1 mM H2O2 for 1 h decreased the integrated intensity of LTR by 63% (p=0.0051) and the lysosomal area by 61% (p=0.0150). Treating erythocytes with the antioxidants GEE, NAC, and HT prior to H2S exposure did not prevent the loss of lysosomes. Therefore, contrary to our hypothesis, antioxidants and iron chelators did not limit lysosomal destabilization. Thus, we conclude that G. dibranchiata may use other mechanism to protect erythrocytes from oxidative stress. University of Florida Collaboration Introduction: Enzymatic digestion of renal calculi into more water-soluble salts could enhance lithotripsy and provide a painless alternative for poor surgical candidates. We performed a preliminary evaluation of the factors affecting enzymatic dissolution rates of the mineral components of calcium oxalate monohydrate (COM), calcium phosphate (CaP), and magnesium ammonium phosphate (struvite) stones. Methods: Oxalate decarboxylase (ODC) from Bacillus subtilis was used to dissolve 25 mg of COM crystals by converting sparsely soluble calcium oxalate to highly soluble calcium formate. Bacterial purine nucleoside phosphorylase (PNP) was used to dissolve 100 mg of CaP and struvite by catalyzing the phosphorolysis of inosine by mineral-derived phosphate. Dissolution of calcium-based crystals was monitored by measuring the increase in solution calcium concentration using a calcium ion selective electrode, while dissolution of struvite crystals was monitored by changes in the mass of insoluble mineral. Results: For COM, a maximum dissolution of 6% was seen, and this did not vary when ODC levels were changed from 50 mg to 200 mg. On addition of the formate-catabolizing enzyme, formate dehydrogenase, dissolution increased to 30%, suggesting that product inhibition by formate limits dissolution of COM crystals by ODC. Phosphate-containing minerals underwent 3% dissolution by 10 units of PNP, corresponding with literature values for the equilibrium constant of the reaction. Conclusions: Product inhibition and equilibrium position are two primary limitations to enzymatic stone dissolution. Both can be overcome by coupling the primary dissolution reactions to other enzymatic reactions whose equilibria favor product formation, reducing product concentration while driving equilibrium towards mineral dissolution.
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2008 Abstracts
