Biochemistry, Biophysics, and Structural Biology Commons^™

Spermine Oxidase (Smo) Activity In Breast Tumor Tissues And Biochemical Analysis Of The Anticancer Spermine Analogues Benspm And Cpenspm, Manuela Cervelli, Gabriella Bellavia, Emiliano Fratini, Roberto Amendola, Fabio Polticelli, Marco Barba, Rodolfo Federico, Fabrizio Signore, Giacomo Gucciardo, Rosalba Grillo, Patrick M. Woster, Robert A. Casero Jr, Paolo Mariottini

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.

Methods

BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT …

Determining The Rate Of Transcription Of T7 Rna Polymerase Using Single Molecule Fluorescence Imaging, Dawn Renee Nichola

Theses, Dissertations and Capstones

It is important to understand the many factors impacting the rate at which an RNA polymerase incorporates nucleotides. The transcription rate of T7 RNA polymerase has been determined using single molecule fluorescence microscopy. A Cy3 labeled circular 45nt ssDNA molecule was used to monitor the transcription process. T7 RNA polymerase was used because it is a single subunit polymerase that does not need any cofactors and will transcribe single-stranded DNA circles that do not contain a promoter. The transcription was monitored by measuring the quasi-periodic change in intensity associated with the transit of the probe through the polymerase as the …

Expression And Characterization Of Antimicrobial Peptides Retrocyclin-101 And Protegrin-1 In Chloroplasts To Control Viral And Bacterial Infections, Baichuan Li

Electronic Theses and Dissertations

Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration …

Electrochemical Sensing In Paper-Based Microfluidic Devices, Zhihong Nie, Christian A. Nijhuis, Jinlong Gong, Xin Chen, Alexander Kumachev, Andres W. Martinez, Max Narovlyansky, George M. Whitesides

Chemistry and Biochemistry

This paper describes the fabrication and the performance of microfluidic paper-based electrochemical sensing devices (we call the microfluidic paper-based electrochemical devices, μPEDs). The μPEDs comprise paper-based microfluidic channels patterned by photolithography or wax printing, and electrodes screen-printed from conducting inks (e.g., carbon or Ag/AgCl). We demonstrated that the μPEDs are capable of quantifying the concentrations of various analytes (e.g., heavy-metal ions and glucose) in aqueous solutions. This low-cost analytical device should be useful for applications in public health, environmental monitoring, and the developing world.

Mechanistic Importance Of Redox Potentials And Conformational Flexibility In Electron-Transferring Flavoproteins, Michael Anthony Swanson

Electronic Theses and Dissertations

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated.

ETF-QO contains one [4Fe-4S]^2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring …

Programmable Diagnostic Devices Made From Paper And Tape, Andres W. Martinez, Scott T. Phillips, Zhihong Nie, Chao-Min Cheng, Emanuel Carrilho, Benjamin J. Wiley, George M. Whitesides

Chemistry and Biochemistry

This paper describes three-dimensional microfluidic paper-based analytical devices (3-D μPADs) that can be programmed (postfabrication) by the user to generate multiple patterns of flow through them. These devices are programmed by pressing single-use ‘on’ buttons, using a stylus or a ballpoint pen. Pressing a button closes a small space (gap) between two vertically aligned microfluidic channels, and allows fluids to wick from one channel to the other. These devices are simple to fabricate, and are made entirely out of paper and double-sided adhesive tape. Programmable devices expand the capabilities of μPADs and provide a simple method for controlling the movement …

Millimeter-Scale Contact Printing Of Aqueous Solutions Using A Stamp Made Out Of Paper And Tape, Chao-Min Cheng, Aaron D. Mazzeo, Jinlong Gong, Andres W. Martinez, Scott T. Phillips, Nina Jain, George M. Whitesides

Chemistry and Biochemistry

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based ―stamp‖) as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimetre. This method provides a new tool to pattern biochemicals—reagents, antigens, proteins, and DNA—on planar substrates. The …

Development Of Ultra-Sensitive Fluorescence Photoamplification Assays For The Detection Of Molecular Recognition Events, Tiffany Priscilla Gustafson

Electronic Theses and Dissertations

During the course of this research a novel method which couples the molecular recognition-triggered photoamplification chain in diaryl ketone adducts of dithiane with a "turn-off" or "turn-on" fluorescence-based assay for the detection of biological targets and ligands, regardless of their nature, through a molecular recognition event has been developed. This research has included several key steps, the most significant being: (1) the design of fluorophore adducts or dyads which recover fluorescence upon photocleavage for a "turn-on" assay and the identification of fluorophores which are quenched upon the photochemical release of a quencher for a "turn off" assay; (2) Optimization of …

Trace Gas And Particle Emissions From Domestic And Industrial Biofuel Use And Garbage Burning In Central Mexico, Ted J. Christian, Robert Yokelson, B. Cardenas, L. T. Molina, G. Engling, S. C. Hsu

Chemistry and Biochemistry Faculty Publications

In central Mexico during the spring of 2007 we measured the initial emissions of 12 gases and the aerosol speciation for elemental and organic carbon (EC, OC), anhydrosugars, Cl(-), NO(3)(-), and 20 metals from 10 cooking fires, four garbage fires, three brick making kilns, three charcoal making kilns, and two crop residue fires. Global biofuel use has been estimated at over 2600 Tg/y. With several simple case studies we show that cooking fires can be a major, or the major, source of several gases and fine particles in developing countries. Insulated cook stoves with chimneys were earlier shown to reduce …

Laboratory Measurements Of Trace Gas Emissions From Biomass Burning Of Fuel Types From The Southeastern And Southwestern United States, I. R. Burling, Robert J. Yokelson, David W. T. Griffith, T. J. Johnson, P. Veres, J. M. Roberts, C. Warneke, S. P. Urbanski, J. Reardon, D. R. Weise, Wei Min Hao, J. De Gouw

Chemistry and Biochemistry Faculty Publications

Vegetation commonly managed by prescribed burning was collected from five southeastern and southwestern US military bases and burned under controlled conditions at the US Forest Service Fire Sciences Laboratory in Missoula, Montana. The smoke emissions were measured with a large suite of state-of-the-art instrumentation including an open-path Fourier transform infrared (OP-FTIR) spectrometer for measurement of gas-phase species. The OP-FTIR detected and quantified 19 gas-phase species in these fires: CO2, CO, CH4, C2H2, C2H4, C3H6, HCHO, HCOOH, CH3OH, CH3COOH, furan, H2O, NO, NO2, HONO, NH3, HCN, HCl, and SO2. Emission factors for these species are presented for each vegetation type burned. …

Evolution Of Trace Gases And Particles Emitted By A Chaparral Fire In California, S. K. Akagi, J. S. Craven, J. W. Taylor, G. R. Mcmeeking, Robert J. Yokelson, I. R. Burling, S. P. Urbanski, C. E. Wold, J. H. Seinfeld, H. Coe, M. J. Alvarado, D. R. Weise

Chemistry and Biochemistry Faculty Publications

Biomass burning (BB) is a major global source of trace gases and particles. Accurately representing the production and evolution of these emissions is an important goal for atmospheric chemical transport models. We measured a suite of gases and aerosols emitted from an 81 hectare prescribed fire in chaparral fuels on the central coast of California, US on 17 November 2009. We also measured physical and chemical changes that occurred in the isolated down-wind plume in the first similar to 4 h after emission. The measurements were carried out onboard a Twin Otter aircraft outfitted with an airborne Fourier transform infrared …

Nip/Duoxa Is Essential For Drosophila Embryonic Development And Regulates Oxidative Stress Response., Xiaojun Xie, Jack Hu, Xiping Liu, Hanjuan Qin, Anthony Percival-Smith, Yong Rao, Shawn S C Li

Biochemistry Publications

NIP/DuoxA, originally cloned as a protein capable of binding to the cell fate determinant Numb in Drosophila, was recently identified as a modulator of reactive oxygen species (ROS) production in mammalian systems. Despite biochemical and cellular studies that link NIP/DuoxA to the generation of ROS through the dual oxidase (Duox) enzyme, the in vivo function of NIP/DuoxA has not been characterized to date. Here we report a genetic and functional characterization of nip in Drosophila melanogaster. We show that nip is essential for Drosophila development as nip null mutants die at the 1(st) larval instar. Expression of UAS-nip, but not …

Meiotic Dna Re-Replication And The Recombination Checkpoint, Nicole Ann Najor

Wayne State University Dissertations

Progression through meiosis occurs through a strict sequence of events, so that one round of DNA replication precedes programmed recombination and two nuclear divisions. Cyclin dependent kinase 1 (Cdk1) is required for meiosis, and any disruption in its activity leads to meiotic defects. The Cdk1 inhibitor, Sic1, regulates the G1-S transition in the mitotic cell cycle and the analogous transition in meiosis. We have employed a form of Sic1, Sic1deltaP^HA, that is mutated at multiple phosphorylation sites and resistant to degradation. Meiosis specific expression of Sic1deltaP^HA disrupts Cdk1 activity and leads to significant accumulation of over replicated …

Single Molecule Studies Of Spliceosomal Snrnas U2-U6, Zhuojun Guo

Wayne State University Dissertations

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging

from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5

and U6) involved in substrate positioning and catalysis. It has been postulated, but never

shown experimentally, that the U2-U6 complex adopts at least two conformations that

reflect different activation states. We have used single-molecule fluorescence to probe the

structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and

mutants of highly conserved regions of U2-U6. Our data show the presence of at least

three distinct conformations in equilibrium. The minimal folding pathway consists of …

Molecular Mechanisms Of Pressure-Stimulated Cancer Cell Signaling, Christina Downey

Wayne State University Dissertations

ABSTRACT

MOLECULAR MECHANISMS OF PRESSURE-STIMULATED CANCER CELL SIGNALING

by

CHRISTINA DOWNEY

June 2010

Advisor: Dr. Marc Basson, MD, PhD

Major: Cancer Biology

Degree: Doctor of Philosophy

Increased extracellular pressure stimulates cancer cell adhesion by a mechanism that is dependent upon beta-1-integrin activation, an intact cytoskeleton, and FAK and Src activation. By a different mechanism, increased extracellular pressure modulates cancer cell proliferation in a manner that is regulated by protein kinase C, but not Src or an intact cytoskeleton. Previous studies from our laboratory have shown that paxillin is a necessary mediator in the pathway by which pressure stimulates adhesion; however, …

Mechanism Of Catch Force: Tethering Of Thick And Thin Filaments By Twitchin., Thomas M Butler, Marion J Siegman

Department of Molecular Physiology and Biophysics Faculty Papers

Catch is a mechanical state occurring in some invertebrate smooth muscles characterized by high force maintenance and resistance to stretch during extremely slow relaxation. During catch, intracellular calcium is near basal concentration and myosin crossbridge cyctng rate is extremely slow. Catch force is relaxed by a protein kinase A-mediated phosphorylation of sites near the N- and C- temini of the minititin twitchin (approximately 526 kDa). Some catch force maintenance car also occur together with cycling myosin crossbridges at submaximal calcium concentrations, but not when the muscle is maximally activated. Additionally, the link responsible for catch can adjust during shortening of …

Comparing Models Of Evolution For Ordered And Disordered Proteins, Celeste J. Brown, Audra K. Johnson, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Most models of protein evolution are based upon proteins that form relatively rigid 3D structures. A significant fraction of proteins, the so-called disordered proteins, do not form rigid 3D structures and sample a broad conformational ensemble. Disordered proteins do not typically maintain long-range interactions, so the constraints on their evolution should be different than ordered proteins. To test this hypothesis, we developed and compared models of evolution for disordered and ordered proteins. Substitution matrices were constructed using the sequences of putative homologs for sets of experimentally characterized disordered and ordered proteins. Separate matrices, at three levels of sequence similarity ( …

The Role Of Igf-1 And Notch Signaling In Thoracic Malignancies., Sandra Eliasz

Dissertations

Thoracic malignancies are one of the deadliest of all cancers, being the leading cause of cancer death in the Western world. Thoracic malignancies arise from different tissues; however the most common are of epithelial (commonly referred to as non-small cell lung cancer, or NSCLC), neuroendocrine (small cell lung cancer, or SCLC) and mesothelial origin (malignant mesothelioma, or MM). The DNA oncogenic virus Simian Virus 40 (SV40) has been shown to cooperate with environmental oncogenic fibers in the onset of MM. Insulin like growth factor-1 (IGF-1) signaling plays a central role in all thoracic malignancies and in the process of SV40-mediated …

New Cyclophanes As Supramolecular Scaffolds: The Synthesis Of Tribenzo-1,4,7-Triazacyclononatriene., Andria M. Panagopoulos

Dissertations

Supramolecular chemistry involves the formation of complex molecular entities that have the capacity to participate in specific molecular recognition of guest molecules. A commonly employed scaffold in supramolecular chemistry is the trimeric crown-shaped molecule cyclotriveratrylene (CTV). CTV has been studied extensively for its capability of binding a number of smaller organic and organometallic guests within its bowl-shaped cleft and has been used as a building block enabling the construction of more complex cryptophanes. The goal of this research is the synthesis and characterization of a novel cyclophane, tribenzo-1,4,7-triazacyclononene and derivatives thereof. These new cyclophanes should have greater versatility than the …

Animal Models Of Alzheimer's Disease, Gemma Casadesus, Gary Arendash, Frank Laferla, Mike Mcdonald

Molecular Biosciences Faculty Publications

No abstract provided.