Biochemistry, Biophysics, and Structural Biology Commons^™

Cryo-Em Structure Of The Archaeal 50s Ribosomal Subunit In Complex With Initiation Factor 6 And Implications For Ribosome Evolution, Basil J. Greber, Daniel Boehringer, Vlatka Godinic-Mikulcic, Ana Crnkovic, Michael Ibba, Ivana Weygand-Durasevic, Nenad Ban

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The …

The Role Of Tumor Suppressors, Ship And Rb, In Immune Suppressive Cells, Michelle Marie Collazo Ruiz

USF Tampa Graduate Theses and Dissertations

Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) have been extensively studied in the past 30-40 years. Their potent suppressive capacity shown in several pathological and clinical settings, such as cancer and transplantation, has made it evident that better understanding their development and function is critical.

Specifically, Tregs play a pivotal role in preventing autoimmunity, graft-versus-host disease (GvHD), and organ graft rejection. We previously demonstrated that germline or induced SH2 domain-containing inositol 5-phosphatase (SHIP) deficiency in the host abrogates GvHD. Here we show that SHIP-deficiency promotes an increase of FoxP3+ cells in both the CD4+CD25+ and the CD4+CD25- T …

Roles Of Trna In Cell Wall Biosynthesis, Kiley Dare, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl‐transfer ribonucleic acid (aa‐tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl‐phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize …

The Role Of Ezh2 In The Regulation Of The Activity Of Matrix Metalloproteinases In Prostate Cancer Cells., Yong Jae Shin, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMPgenes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation …

The Mechanical Properties Of Epoxy Composites Filled With Rubbery Copolymer Grafted Sio2, Jianing Gao, Junting Li, Brian C. Benicewicz, Su Zhao, Henrik Hillborg, Linda S. Schadler

Faculty Publications

This study demonstrated a method for toughening a highly crosslinked anhydride cured DGEBA epoxy using rubbery block copolymer grafted SiO2 nanoparticles. The particles were synthesized by a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization. The inner rubbery block poly(n-hexyl methacrylate) (PHMA) had a glass transition temperature below room temperature. The outer block poly(glycidyl methacrylate) (PGMA) was matrix compatible. A rubbery interlayer thickness of 100% and 200% of the particle core radius was achieved by grafting a 20 kg/mol and a 40 kg/mol PHMA at a graft density of 0.7 chains/nm² from the SiO₂ surface. The 20 kg/mol rubbery …

Structural Basis For Activation Of Calcineurin By Calmodulin, Julie Rumi-Masante, Farai I. Rusinga, Terrence E. Lester, Tori B. Dunlap, Todd D. Williams, A. Keith Dunker, David D. Weis, Trevor P. Creamer

Center for Structural Biology Faculty Publications

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured …

Usp8 Promotes Smoothened Signaling By Preventing Its Ubiquitination And Changing Its Subcellular Localization, Ruohan Xia, Hongge Jia, Junkai Fan, Yajuan Liu, Jianhang Jia

Molecular and Cellular Biochemistry Faculty Publications

The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that …

Aquaporin 4 Is A Ubiquitously Expressed Isoform In The Dogfish (Squalus Acanthias) Shark, Christopher P. Cutler, Bryce Maciver, Gordon Cramb, Mark L. Zeidel

Biology: Faculty Publications

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further …

Fully Enclosed Microfluidic Paper-Based Analytical Devices, Kevin M. Schilling, Anna L. Lepore, Jason A. Kurian, Andres W. Martinez

Chemistry and Biochemistry

This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.

[Accepted Article Manuscript Version (Postprint)] Plant Peroxisomes: Biogenesis And Function, Jianping Hu, Alison Baker, Bonnie Bartel, Nicole Linka, Robert Mullen, Sigrun Reumann, Bethany Zolman

Biology Department Faculty Works

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of …

"Fine-Tuning" Of Ribosomal Structure And Functions By Pseudouridylation And Rna-Protein Interactions, Jun Jiang

Wayne State University Dissertations

ABSTRACT

"Fine-tuning" of ribosomal structure and functions by pseudouridylation and RNA-protein interactions

by

JUN JIANG

AUGUST 2012

Advisor: Prof. John SantaLucia Jr.

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Ribosomal structure and functions appear to be "fine-tuned" by pseudouridylation and RNA-protein interactions. Pseudouridylation may promote base stacking interactions by mediating the base stacking between residues on both sides. In the RNA duplex region, this enhanced stacking interaction contributes to stabilization of duplex folding. In the loop region, enhanced stacking in one structural motif may destabilize the conformation of adjacent structural residues. This hypothesis is supported by both UV-melting experiments, where …

Conformational Properties Of Cardiolipin-Bound Cytochrome C, Jonas Hanske, Jason R. Toffey, Anna M. Morenz, Amber J. Bonilla, Katherine H. Schiavoni;, Ekaterina V. Pletneva

Dartmouth Scholarship

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions P(r) …

Domain Architecture Of A Calcium-Permeable Ampa Receptor In A Ligand-Free Conformation, Charles R. Midgett, Avinash Gill, Dean R. Madden

Dartmouth Scholarship

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves …

Increased Variability Of Microbial Communities In Restored Salt Marshes Nearly 30 Years After Tidal Flow Restoration, Anne E. Bernhard, David Marshall, Lazaros Yiannos

Biology Faculty Publications

We analyzed microbial diversity and community composition from four salt marsh sites that were impounded for 40–50 years and subsequently restored and four unimpounded sites in southeastern Connecticut over one growing season. Community composition and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) and sequence analysis of 16S ribosomal RNA (rRNA) genes. Our results indicated diverse communities, with sequences representing 14 different bacterial divisions. Proteobacteria, Bacteroidetes, and Planctomycetes dominated clone libraries from both restored and unimpounded sites. Multivariate analysis of the TRFLP data suggest significant site, sample date, and restoration status effects, but the exact causes of these …

Changing Water Quality In Great Pond: The Roles Of Lake Sediments, Invasive Macrophytes, And The Watershed, Colby Environmental Assessment Team, Colby College, Problems In Environmental Science Course (Biology 493), Colby College

Colby College Watershed Study: Great Pond (2012, 2010, 1998)

Eutrophication as a result of human activity is a threat to lake water quality globally and within the state of Maine. Great Pond, in the Belgrade Lakes region of Maine, has traditionally been an oligotrophic lake that is experiencing early signs of eutrophication and is currently classified as a mesotrophic lake. In the fall of 2012, the Colby Environmental Assessment Team (CEAT) measured the primary sources of nutrient loading to Great Pond including the catchment and the lake sediment, current water quality in Great Pond, and the potential impact of the variable milfoil invasion on the lake’s water quality. An …

The Role Of Ezh2 In The Regulation Of The Activity Of Matrix Metalloproteinases In Prostate Cancer Cells., Yong Jae Shin, Jeong-Ho Kim

Biochemistry and Molecular Medicine Faculty Publications

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation …

Two-Dimensional Blood Flow Velocity Estimation Using Apparent Speckle Pattern Angle Dependence On Scan Velocity, Tiantian Xu, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An algorithm which measures the lateral component of blood flow velocity was developed in our previous studies based on the increase in speckle size due to relative motion between moving scatterers and spatial rate of scanner A-line acquisition (scan velocity). In this paper, the apparent dominant angle of the speckle pattern in a straight vessel was investigated and a new method of two-dimensional blood flow velocity estimation is introduced. Different scan velocities were used for data acquisition from blood flow traveling at an angle relative to the ultrasound beam. The apparent angle of the speckle pattern changes with different scan …

Monitoring Cerebral Hemodynamics With Transcranial Doppler Ultrasound During Cognitive And Exercise Testing In Adults Following Unilateral Stroke, Brian P. Watt, Judith M. Burnfield, Edward J. Truemper, Thad W. Buster, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An observational study was performed as a preliminary investigation into the use of transcranial Doppler ultrasound (TCD) for recording cerebral hemodynamic changes during multiple tasks. TCD is a method of measuring cerebral blood flow (CBF) using ultrasound transducers in contact with the surface of the head. Using the maximum flow envelope of the Doppler spectrum returning from the middle cerebral artery (MCA), standard clinical flow indices can be calculated and displayed in real time providing information concerning perturbations in CBF and their potential cause. These indices as well as flow velocity measurements have been recognized as useful in measuring changes …

Toxicological Assessment Of Intra‐Tracheal Instilled Nanoceria On Kidneys Of Sprague‐Dawley Rats, Geeta Nandyala

Theses, Dissertations and Capstones

Recent studies suggest that use of nanoceria in industry is associated with increased risk of human and environmental exposure. How inhaled nanoceria might affect the kidneys is not yet known. To investigate this possibility, Sprague Dawley rats were exposed to a single dose of 7mg/kg body weight cerium oxide nanoparticles by intratracheal instillation. Animals were sacrificed at day 1, 3, 14, or 28 days after exposure and the kidneys collected for histological and biochemical analysis. No significant difference was observed in kidney to body weight ratio between the different groups. Hematoxylin and eosin staining did not reveal any significant changes …

Diversity Of The Ohio River Bacterial Communities Using Next-Generation Sequencing Techniques, Emily Michele Anneken

Theses, Dissertations and Capstones

The ability to sequence bacterial genetic material directly from environmental samples has unlocked the field of metagenomics. Next-generation sequencing techniques, such as pyrosequencing, have been instrumental in providing knowledge about bacterial communities. This study focused on identifying the bacterial diversity of the Ohio River via pyrosequencing and compared the diversity of cultivable bacteria versus the entire river community. To ensure the maximum number of cultivable bacteria was obtained, cultures were grown on nonselective media and incubated at river temperature. Bacterial DNA was extracted, sequenced, and classified. The dominant phyla for the Ohio River included Cyanobacteria (38-66% of the total), Actinobacteria …