Life Sciences Commons^™

Mechanism Of Action Studies On A New Class Of Anticancer Nucleosides, Megan E. Browning

Theses and Dissertations

We have completed mechanism of action studies on a new class of anticancer nucleosides typified by a novel nucleoside discovered in our lab, MAP-870. In order to study the mechanism of MAP-870, several experiments were completed on a colorectal adenocarcinoma cell line, HT-29, including trypan blue cell count, sulforhodamine B assays, flow cytometry of cell cycle, propidium iodide incorporation, and phosphatidylserine externalization, Caspase-Glo3/7 assays, DNA fragmentation gel, cyclophilin A release gel, PAMPA, and confocal imaging. Sulforhodamine B assays show that MAP-870 does indeed cause growth inhibition and cell death in the model tested. PAMPA assays show that MAP-870 does not …

Effect Of Paired Apertures In A Periodic Hole Array On Higher Order Plasmon Modes, Fang-Tzu Chuang

Fang-Tzu Chuang

We demonstrate that the transmission of higherorder surface plasmon modes in the mid-infrared range can be enhanced through rectangular hole array on the basis of paired apertures. Experiments prove that enhanced high-order transmission can be generated by either identical shapes or combinations of different hole shapes in pairs. The structure factor is adopted to explain the observed intensity of enhanced transmission. Numerical simulations of the enhanced secondorder mode verify a significant field enhancement in a unit cell of pairs. It is clarified that the separation between the paired apertures and the paired resonance is the key to determine certain higher-order …

Phylogenetic Engineering Of The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large Subunit In Chlamydomonas Reinhardtii, Boon Hoe Lim

Department of Biochemistry: Dissertations, Theses, and Student Research

Thirty-four residues in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) may account for the kinetic differences between Rubisco enzyme from green algae and land plants. By substituting these "phylogenetic residues" as groups and combinations of groups in the large subunit of the green alga Chlamydomonas reinhardtii with those of land-plant Rubisco, the functions and relationships of these "phylogenetic groups" were determined.

A phylogenetic-group substitution at the base of catalytic loop 6 of the large subunit decreases the CO₂/O₂ specificity of the enzyme, but function is restored by a further phylogenetic-group substitution at the carboxy-terminal tail. Therefore, these …

Differential Effects Of The Hydrophobic Surfactant Proteins On The Formation Of Inverse Bicontinuous Cubic Phases, Mariya Chavarha, Ryan W. Loney, Kamlesh Kumar, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce formation of the bicontinuous structures and faster adsorption by similar mechanisms, then they should also have differential ability to form the cubic phases. To test this hypothesis, we measured small angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the doserelated …

Cross Regulation Of Sirtuin 1, Ampk, And Ppary In Conjugated Linoleic Acid Treated Adipocytes, Shan Jiang, Wei Wang, Jess Miner, Michael E. Fromm

Department of Biochemistry: Faculty Publications

Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) reduces triglyceride (TG) levels in adipocytes through multiple pathways, with AMP-activated protein kinase (AMPK) generally facilitating, and peroxisome proliferator-activated receptor γ (PPARγ) generally opposing these reductions. Sirtuin 1 (SIRT1), a histone/protein deacetylase that affects energy homeostasis, often functions coordinately with AMPK, and is capable of binding to PPARγ, thereby inhibiting its activity. This study investigated the role of SIRT1 in the response of 3T3-L1 adipocytes to t10c12 CLA by testing the following hypotheses: 1) SIRT1 is functionally required for robust TG reduction; and 2) SIRT1, …

Ivermectin Inhibits Growth Of Chlamydia Trachomatis In Epithelial Cells, Matthew A. Pettengill, Verissa W. Lam, Ikechukwu Ollawa, Camila Marques-Da-Silva, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia.

Development And Validation Of A Simple Method For The Detection Of Fascaplysin In Plasma, Kenneth H. Swartz, Frederick A. Valeriote, Joseph Media, Tyler A. Johnson, Karen Tenney, Phillip Crews, Jiajiu Shaw

Natural Sciences and Mathematics | Faculty Scholarship

Fascaplysin is a cytotoxic natural product isolated from a variety of Indo-Pacific marine organisms, primarily Fascaplysinopsis sponges and Didemnum tunicates. Positive xenograft studies involving this alkaloid structural class have indicated that fascaplysin may serve as an important lead compound for preclinical development. This study was undertaken as a prelude to a full pharmacokinetics and therapeutic assessment of fascaplysin. We describe here a simple plasma preparation and a rapid HPLC method for the detection of fascaplysin in mice. The method was validated by parameters including good linear correlation, a limit of quantification of 107.1 μg/ mL, and a good precision with …

Effects Of Canola And Corn Oil Mimetic On Jurkat Cells, Gabriela Ion, Kayla Fazio, Juliana A. Akinsete, W. Elaine Hardman

Gabriela Ion

BACKGROUND: The Western diet is high in omega-6 fatty acids and low in omega-3 fatty acids. Canola oil contains a healthier omega 3 to omega 6 ratio than corn oil. Jurkat T leukemia cells were treated with free fatty acids mixtures in ratios mimicking that found in commercially available canola oil (7% α-linolenic, 30% linoleic, 54% oleic) or corn oil (59% linoleic, 24% oleic) to determine the cell survival or cell death and changes in expression levels of inflammatory cytokines and receptors following oil treatment. METHODS: Fatty acid uptake was assessed by gas chromatography. Cell survival and cell death were …

Maternal Consumption Of Canola Oil Suppressed Mammary Gland Tumorigenesis In C3(1) Tag Mice Offspring, Gabriela Ion, Juliana A. Akinsete, W. Elaine Hardman

Gabriela Ion

Background: Maternal consumption of a diet high in omega 6 polyunsaturated fats (n-6 PUFA) has been shown to increase risk whereas a diet high in omega 3 polyunsaturated fats (n-3 PUFA) from fish oil has been shown to decrease risk for mammary gland cancer in female offspring of rats. The aim of this study was to determine whether increasing n-3 PUFA and reducing n-6 PUFA by using canola oil instead of corn oil in the maternal diet might reduce the risk for breast cancer in female offspring. Methods: Female SV 129 mice were divided into two groups and placed on …

Consumption Of High Ω-3 Fatty Acid Diet Suppressed Prostate Tumorigenesis In C3(1) Tag Mice, Juliana Akinsete, Gabriela Ion, Theodore Witte, W. Hardman

Gabriela Ion

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to …

(1)H, (15)N And (13)C Backbone Resonance Assignments Of The Tpr1 And Tpr2a Domains Of Mouse Sti1., Andrzej Maciejewski, Marco A Prado, Wing-Yiu Choy

Biochemistry Publications

Hop/STI1 (Hsp-organizing protein/stress-induced-phosphoprotein 1) is a molecular co-chaperone, which coordinates Hsp70 and Hsp90 activity during client protein folding through interactions with its TPR1 and TPR2A domains. Hsp90 substrates include a diverse set of proteins, many of which have been implicated in tumorigenesis. Over-expression of Hsp90 in cancer cells stabilizes mutant oncoproteins promoting cancer cell survival. Disruption of Hsp90 and its co-chaperone machinery has become a promising strategy for the treatment of cancer. STI1 has also been described as a neurotrophic signaling molecule through its interactions with the prion protein (PrP(C)). Here, we report the (1)H, (13)C and (15)N backbone assignments …

Expression Of Fused In Sarcoma Mutations In Mice Recapitulates The Neuropathology Of Fus Proteinopathies And Provides Insight Into Disease Pathogenesis, Christophe Verbeeck, Mariely Dejesus-Hernandez, Carolina Ceballos-Diaz, Jannet Kocerha, Todd Golde, Pritam Das, Rosa Rademakers, Dennis W. Dickson, Thomas Kukar

Jannet Kocerha

Background: Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease …

Comparison Of Phenolic Compounds And Antioxidant Capacities Of Traditional Sorghum Beers With Other Alcoholic Beverages, Fatouma Abdoul-Latif, Romaric G. Bayili, Louis C. Obame, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Thirty samples of sorghum beers “dolo” were selected from traditionally fermented household manufacturers from Burkina Faso. Dolo samples were screened for their total phenolic content, proanthocyanidins and putative antioxidant capacities, and were compared with industrial beers and wines. Total phenols were measured using the Folin-Ciocalteu method. Proanthocyanidins content were determined by the method of HCl-butanol hydrolysis. Antioxidant activities were evaluated both with 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and by the trolox equivalent antioxidant capacity (TEAC) using 2,2’-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid radical) (ABTS•+). The average contents of total phenols and proanthocyanidins were 506 μg GAE/ml of dolo and 45 μg APE/ml of dolo, respectively. An …

Characterization Of The Monomer-Dimer Equilibrium Of Recombinant Histo-Aspartic Protease From Plasmodium Falciparum, Huogen Xiao, Lee-Ann Briere, Stanley Dunn, Rickey Yada

Stanley D Dunn

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx-tHAP. The present study found that the recombinant Trx-tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.2% CHAPS. Trx-tHAP fusion protein was processed into a mature form of tHAP (mtHAP) by both autoactivation, and activation with either enterokinase or plasmepsin II. Using gel filtration chromatography as well as sedimentation velocity and equilibrium ultracentrifugation, …

The B Subunits In The Peripheral Stalk Of F1f0 Atp Synthase Preferentially Adopt An Offset Relationship, Shane Claggett, Mac O'Neil Plancher, Stanley Dunn, Brian Cain

Stanley D Dunn

The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b' subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., …

Functionally Compensating Coevolving Positions Are Neither Homoplasic Nor Conserved In Clades, Gregory Gloor, Gaurav Tyagi, Dana Abrassart, Andrew Kingston, Andrew Fernandes, Stanley Dunn, Christopher Brandl

Stanley D Dunn

We demonstrated that a pair of positions in phosphoglycerate kinase that score highly by three nonparametric covariation measures are important for function even though the positions can be occupied by aliphatic, aromatic, or charged residues. Examination of these pairs suggested that the majority of the covariation scores could be explained by within-clade conservation. However, an analysis of diversity showed that the conservation within clades of covarying pairs was indistinguishable from pairs of positions that do not covary, thus ruling out both clade conservation and extensive homoplasy as means to identify covarying positions. Mutagenesis showed that the residues in the covarying …

36 Degrees Step Size Of Proton-Driven C-Ring Rotation In Fof1-Atp Synthase, Monika Düser, Nawid Zarrabi, Daniel Cipriano, Stefan Ernst, Gary Glick, Stanley Dunn, Michael Börsch

Stanley D Dunn

Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

Elastic Deformations Of The Rotary Double Motor Of Single F(O)F(1)-Atp Synthases Detected In Real Time By Förster Resonance Energy Transfer., Stefan Ernst, Monika G Düser, Nawid Zarrabi, Stanley D Dunn, Michael Börsch

Biochemistry Publications

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to …

Two Infrared Emission Modes With Different Wavelengths And Orthogonal Polarization In A Waveguide Thermal Emitter, Fang-Tzu Chuang

Fang-Tzu Chuang

This study investigates a Au/SiO2/Au waveguide thermal emitter incorporating a metallic grating embedded in the SiO2 layer. The metal grating acts as a beam splitter, dividing the device into two waveguide structures determined by the polarization of the waveguide modes. The thermal radiation spectrum exhibits two peaks with orthogonal polarization. The emitted wavelengths of the two waveguide modes can be adjusted by controlling the thickness of the SiO2 layers on both sides of the metallic grating during the fabrication process. The emission peaks have ratios of the full width at half maximum to the peak wavelength of 0.057 and 0.05 …

Functions Of The Arabidopsis Kinesin Superfamily Of Microtubule-Based Motor Proteins, Chuanmei Zhu, Ram Dixit

Biology Faculty Publications & Presentations

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. …

Elastic Deformations Of The Rotary Double Motor Of Single F(O)F(1)-Atp Synthases Detected In Real Time By Förster Resonance Energy Transfer., Stefan Ernst, Monika G Düser, Nawid Zarrabi, Stanley D Dunn, Michael Börsch

Biochemistry Publications

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to …

Minireview: Protein Interactions, Jessica Child

Honors Theses and Capstones

No abstract provided.

The Contribution Of Oxidative Stress In The Protein Damage And Dna Lesion In Alzheimer's Disease Neuropathology, Cheng Zhang

Doctoral Dissertations

Glutathione (GSH) plays an essential role in the intracellular antioxidant defense against the oxidant radicals, especially the ·OH radical. To understand the early and progressive cellular changes in Alzheimer's disease (AD) development, we investigated reduced glutathione/oxidized glutathione (GSH/GSSG) status in a double mutated AD transgenic mouse model (B6.Cg-Tg), which carries Swedish amyloid precursor protein mutation (APPswe) and exon 9 deletion of the PSEN1 gene. Likewise, S-glutathionylation (Pr-SSG) is a specific post-translational modification (PTM) of cysteine residues by the addition of glutathione. S-glutathionylated proteins induced by oxidative stress play an essential role in understanding the pathogenesis of the aging …

Patterns Of Chemosensory Behavior In A Closed Population Of Wild African Elephants (Loxodonta Africana), Russell W. Blogg

Electronic Theses and Dissertations

Chemosensory behaviors are used by many mammalian species to assess chemical signals in the environment. These chemical signals may contain important information about reproductive state, identity, status, or location of conspecifics. Elephants are a long-lived species and males reproduce at a much later age than females, which provides a protracted developmental period for males. This study examined chemosensory behaviors in a population of African elephants living in Addo Elephant National Park, South Africa and demonstrated that patterns emerge as elephants develop and approach sexual maturity. Older pubescent males (15-19 year olds) performed more chemosensory behaviors than younger pubescent males (10-14 …

Re-Introduction Of Transmembrane Serine Residues Reduce The Minimum Pore Diameter Of Channelrhodopsin-2, Robert Dempski, Ryan Richards

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but …

N-Terminal Domain Of Vacuolar Snare Vam7p Promotes Trans-Snare Complex Assembly, Hao Xu, William T. Wickner

Dartmouth Scholarship

SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its …

Exploring The Role Of A Conserved Class A Residue In The Ω-Loop Of Kpc-2 Β-Lactamase: A Mechanism For Ceftazidime Hydrolysis

Chemistry Faculty Publications

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we …

Serotyping Group B Streptococci In A Small Community Hospital: An Analysis Of Distribution And Site Of Isolation, Jennifer M. Smith, Jason A. Rexroth, David G. Chaffin, Susan H. Jackman

Susan H. Jackman

Objective: To determine the prevalence and site of isolation of different serotypes of group B streptococcus (GBS) colonization or infection at a small community hospital. Methods: GBS isolates were obtained from a small community hospital and were then serotyped as la, Ib, II, III, IV, V or non-typeable. Hospital records were reviewed for patient sex, age and pregnancy status as well as the site of GBS isolation. Results: GBS serotypes Ia, III and V were most common and accounted for over 60% of the total number of isolates. Serotype Ia was most prevalent in reproductive-age females, while serotypes V and …

Murine Epidermal Cell Antigen (Skn)-Directed Autoimmunity Induced By Transfer Of Cd4+ T Cells, Susan H. Jackman, Shivaleela Keerthy, Giselle Perry

Susan H. Jackman

While pathogenic T cells have been identified for several diseases with epithelial cell damage, an autoimmune T cell-mediated response targeted against a known keratinocyte antigen has not been reported. Previously we described an autoimmune response directed to the mouse epidermal cell antigens, Skn. For our murine model, primed Skn-immune lymphocytes are adoptively transferred to recipients, which develop lesions at the site of mild skin trauma. In this study we investigated the nature of the autoimmune component of the Skn response. A time-course study demonstrated a relationship between the number of primed Sknimmune cells injected and the severity of skin lesions …

Purification And Characterization Of Novel Nucleases From A Thermophilic Fungus, Kyle Landry

Kyle S Landry

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 ^oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined …