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However, further increases in sensitivity are needed to render NMR even more powerful under mild, physiologically relevant conditions. Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) is a laser-driven method that utilizes the radical pair that forms between oxidizable amino acids and photo-excited dyes. This method is capable of leading to large increases in NMR sensitivity in solution. The application of photo-CIDNP to 13C nuclei in conjunction with 13C-1H heteronuclear correlation has proven particularly successful for the highly sensitive detection of aromatic amino acids in liquid state NMR. However, combining 13C photo-CIDNP with 1H-13C-1H heteronuclear correlation has the potential of yielding even further sensitivity enhancements. Therefore, we have combined photoCIDNP with 1H-13C-1H heteronuclear correlation to seek such enhancements. While the EPIC-HSQC pulse sequence exploits 1H-15N-1H correlation, the novel sequence that we designed, denoted as 13C-EPIC-HSQC, focuses on 1H-13CH correlation. Theory predicts that the novel NMR pulse sequence should provide a four-fold increase in sensitivity.
1 It is hoped that this enhancement will render solution-state NMR more readily applicable to low-micromolar solutions.
Method development and applications to tryptophan and other biomolecules of medical relevance will be discussed in the poster.
BACTERIAL DYNAMICS IN THE MOSQUITO GUT VISUALIZED BY FLUORESCENTLY LABELLED BACTERIAL
STRAIN OF ENTEROBACTER SPAlejandro Uentillie, Jiannong Xu.
New Mexico State University, Las Cruces, NM.
The mosquito gut is a complex ecosystem filled with microbiomes. The gut content, which has many microbiome fauna, affects the fecundity and immunity of the host’s traits. In order to understand the dynamics of bacterial behavior, we introduced the bacterium Enterobacter sp. Ag1 into mosquito gut by feeding. To be able to track the bacteria, we tagged the bacterial strain with green fluorescent protein (GFP). After feeding mosquitoes on sugar pad with tagged bacteria, we tracked the bacteria in the gut by observing mosquito gut under a fluorescent microscope.
The results showed that tagged bacteria were present in the sugar-fed and proliferated in blood-fed mosquito guts.
To further understand how bacteria colonize the gut and the genes involved in colonization, we generated bacterial mutants. Two mutants were selected to test their capability to colonize the gut. One mutant P1A6 had gene encoding LPS O antigen ligase disrupted. The other mutant P12D2 had gene encoding ADP-heptose:LPS heptosyl transferase I disrupted. Both genes are involved in LPS biosynthesis. The disruption of those genes had effects on LPS structure on the bacterial cell wall. We mixed the RFP tagged wild type (wt) and GFP-tagged mutants in 1:1 ratio, and let mosquitoes ingest them. Then we tracked their dynamics in the gut. Gut dissection revealed more of the wt/RFP than the mutants P12D2 and P1A6. The results indicate that both genes were required for bacterial colonization in the mosquito gut.
NUTRIENT CONCENTRATION AND ITS EFFECTS ON SECONDARY METABOLITE PRODUCTION IN
MYXOBACTERIA CULTURESPeter Gomez, Nicholas Lorig-Roach, Tyler Johnson, Marija DraÂškoviÄ‡, Mitchel Crews, Phil Crews.
University of California, Santa Cruz, Santa Cruz, CA.
Myxobacteria, a group of Gram-negative swarming aerobic bacteria found in terrestrial and marine sediments, possess a largely unexplored potential for discovery of novel organic compounds with useful applications as therapeutic leads. Some strains are known to be producers of useful chemotypes such as myxovirescins, secondary metabolites previously reported to possess antibiotic activity from Myxococcus virescens. However, myxobacteria are often challenging to grow in culture, making it difficult to acquire enough material for chemical analysis to broaden the knowledge base of obtainable secondary metabolites of this organic type. This experiment attempts to better understand the nutritional requirements for optimal myxobacterial metabolite production by comparing three liquid media types with different nutrient concentrations. As a test subject, we shall be using a myxobacteria strain isolated from sediment samples taken from Wilder Ranch State Park, a coastal region in northern California. Initial
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investigations have shown that this strain is a producer of known myxovirescin analogues. We will inoculate three 3 L cultures containing 1X, 1/4X, and 1/8X concentrations of cooked yeast and casitone, the primary nutrients used to feed myxobacteria in our cultures. The produced metabolites will then be adsorbed onto compound-binding resin and extracted with methanol. The extracts will then be chromatographically fractionated and analyzed using LC-MS/ ELSD. By quantitatively comparing myxovirescin analogue production from these three different growth conditions, we hope to increase our understanding of myxobacterial nutritional preference and obtain insight into improving culturing techniques.
LOCALIZATION OF EIF4G1 IN ARABIDOPSIS THALIANAAmy Prater, Karen Browning.
University of Texas at Austin, Austin, TX.
Translation is a highly regulated process involving initiation, elongation, and termination. One of the first steps in initiation is the binding of the eIF4F complex composed of eIF4G, the scaffolding protein, and eIF4E, the capbinding protein, to the 5’ capped region of the mRNA and bringing it into contact with the 40S ribosomal subunit. The mRNA-bound ribosome then scans along the mRNA until it reaches the correct initiation codon. Upon recognition, a conformational change allows for the 60s ribosomal subunit to associate with the complex, permitting the sequence
to be translated into protein. In plants, there is an alternative complex to eIF4F that performs this same function:
eIFiso4F, which is composed of two subunits, eIFiso4E and eIFiso4G. There are two possible isoforms of this iso4F complex, iso4G1 with iso4E or iso4G2 with iso4E due to the presence of two genes encoding iso4G. As opposed to the eIFiso4G1 complex, eIFiso4G2 expression is generally low within the plant. However, when 4G or iso4G1 are knocked out, the presumed low expression levels of eIFiso4G2 are still enough to support plant growth. To aid in understanding the role of the eIFiso4G2 and eIFiso4G1, homozygous eIFiso4G2 and eIFiso4G1 GFP-tagged plants were grown on plates and then examined using confocal microscopy to determine where in the cell, as well as where in the plant, eIFiso4G2 expression is localized.
A STUDY OF THE BEHAVIOR OF VOLTAGE-CURRENT CURVES FOR THE PROTOTYPE INTERFACE FILMBACTERIA / MCD-NCD Barbara Avalos-Cavero1, Javier Avalos2.
Universidad de Puerto Rico, Río Piedras, San Juan, PR, 2Universidad de Puerto Rico, Bayamón, Bayamón, PR.
1 The National Institutes of Health reported that 60% of all microbial infections are caused by biofilms, which are due to microbial presence on the surfaces of implants in the human body and on surgery tools. The medical industry can benefit greatly from coatings designed to reduce bacterial viability on implants and medical tools. In the present work, we have evaluated the response to the electric current generated by a biofilm formed by bacteria that can be either Gram negative or positive on the surface of microcrystalline (MCD) and nanocrystalline (NCD) diamond.
The preliminary results demonstrated a change in the effective resistance of NCD and MCD material when a biofilm is formed on its surface. Furthermore, an oscillating behavior was observed in the curves of electric current versus voltage due to the presence of a bacterial strain droplet on the surface of MCD and NCD starting at 1.6 v and ranging from 0.2 v to 4.0 v, similar to RC electric circuit. These studies are focused on the hypothesis of the mechanism of bacterial inhibition most accepted, which involves an electrostatic relationship between the organism and the substrate in contact. The voltage-current measurements were taken with modified contact angle equipment, elaborated in the laboratory. Other studies of characterization were done using the scanning electron microscope, atomic force microscope, and Raman spectroscopy. These studies allow us to infer that the formation of biofilm on biomedical instruments can alter their electrical response, thus providing misleading results, which can be prejudicial for patient diagnosis.
CIRCULAR DICHROISM FOR MONITORING THE CONFORMATIONAL CHANGES OF AMYLOIDOGENIC ISLET
AMYLOID POLYPEPTIDEBianca Malaspina, David Moffet, Travis Whyte,.
Loyola Marymount University, Los Angeles, CA.
Islet amyloid polypeptide (IAPP) is an amyloidogenic protein secreted in the β cells of the pancreas. Similar to other amyloid proteins, IAPP has the ability to misfold and form amyloid plaques in the β cells, causing cell death and
BIOCHEMICAL AND STRUCTURAL STUDIES OF THE IST1-ULK3 COMPLEXKira M. Miller1, Dawn M. Wenzel2, Jack J. Skalicky2 Wesley I. Sundquist2.
Whitworth University, Spokane, WA, 2University of Utah, Salt Lake City, UT.
1 The ESCRT pathway mediates membrane fission during the abscission step of cytokinesis. ESCRT-III proteins, including IST1, play important roles during abscission, including membrane constriction and recruitment of other proteins. We have recently shown that the kinase ULK3 interacts directly with IST1 and that depletion of ULK3 induces abscission defects, implying that the two proteins work together during abscission. We are now characterizing how IST1 and ULK3 interact. ULK3 contains two predicted MIT (microtubule interacting and transport) domains which are 3-helix bundles that bind MIMs (MIT interaction motifs) within ESCRT-III proteins. We hypothesize that one or both of the ULK3 MIT domains bind one or both of the two MIM elements within IST1. To map the ULK3-IST1 binding sites, we will compare IST1 binding to wild-type ULK3 vs. ULK3 proteins with inactivating point mutations in either or both of the MIT domains. We are also using NMR spectroscopy to characterize the binding of ULK3 to IST1. Specifically, we have purified a 15N-labeled ULK3 construct that spans both MIT domains (termed ULK3(MIT)2) and used chemical shift changes to follow the binding of an unlabeled fragment of IST1 (termed IST1(MIM)). Backbone amide resonances in ULK3(MIT)2 shift upon titration of IST1(MIM), confirming that the ULK3-IST1 interaction is direct. NMR spectroscopy will now be used to determine the three dimensional structure of the IST1(MIM)-ULK3(MIT)2 complex.
This structure will reveal how IST1 and ULK3 interact, help us to understand IST1 regulation, and possibly guide the design of new strategies to prevent or repair cytokinesis defects.
QUANTIFYING ANTIOXIDANT ABILITY OF IONIC MANGANESE IN WORM-FREE C. ELEGANS EXTRACTS BY
ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPYKatherina Chua, Chandra Srinivasan, Michael Bridges.
California State University, Fullerton, Fullerton, CA.
While superoxide dismutase and other antioxidant enzymes combat the normal production of free radicals within a cell. An excess of free radicals has been linked to pro-oxidant effects that can lead to cellular damage, promoting aging and various degenerative diseases. Divalent manganese, Mn(II), has been shown to function as a protective factor against free radicals in both prokaryotic and eukaryotic organisms. However, the mechanism by which ionic manganese plays a role in scavenging free radicals is not yet understood. In order to study this mechanism, Caenorhabditis elegans is used as a multicellular model organism, with a genome approximately 40% homologous to that of humans, to study ionic manganese and its activity with transient, unstable reactive species in vivo. By nitrone spin trapping the most reactive intracellular radicals, including hydroxyl radical (OH), we will be able to investigate free radical scavenging ability of divalent manganese within C. elegans for the first time via electron paramagnetic resonance spectroscopy. Preliminary control experiments have shown slight reduction of radical signal in the presence of unbound, free divalent manganese. Although these results do not significantly confirm antioxidant effects, these control experiments do not account for the potential effects of small cellular molecules that may improve the antioxidant ability of ionic manganese. Now, our primary goal is to examine antioxidant ability in vitro in cell-free low molecular weight C. elegans worm extracts in the absence and presence of ionic manganese.
TOWARDS UNDERSTANDING THE FUNCTIONAL CONFORMATION OF THE GRB7 PROTEINDarius Warren, Barbara Lyons.
New Mexico State University, Las Cruces, NM.
Growth factor receptor-bound protein 7 (Grb7) plays a role in cell migration pathways mediated by integrins and focal adhesion kinase (FAK). In previous research, Porter et al. found the dimerization disassociation constants of full length Grb7 and the Grb7-SH2 domain alone were the same order of magnitude (11 mM and 22 mM, respectively).
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This suggests the Grb7-SH2 domain forms the primary basis for dimerization in the Grb7 protein. Subsequently, Depetris et al. solved the crystal structure of the Grb10 RA-PH protein domains. Grb10 is a homologous protein to Grb7. In this structure, and through size exclusion data, there is some indication the RA-PH domains of the Grb10 protein also form a dimer. Further, mutation of two residues in the C-terminal a-helix of the PH domain may cause disruption of the dimer interface, and subsequent loss of RA-PH domain association. In our own laboratory we have found, through size exclusion chromatography, the Grb7-RA domain also exists as a dimer. Our own result, and that of Depetris et al., contradicts the Porter et al. findings describing the basis of dimerization in the Grb7 protein.
We believe the Grb7-RA domain may play an important role in homodimerization of the full-length Grb7 protein.