Biochemistry, Biophysics, and Structural Biology Commons^™

Composition And Evolution Of The Vertebrate And Mammalian Selenoproteomes, Marco Mariotti, Perry G. Ridge, Yan Zhang, Alexei V. Lobanov, Thomas H. Pringle, Roderic Guigo, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Background: Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24.

Methodology/Principal Findings: We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian …

Critical Heat Stress Evaluation Of Two-Layer Clothing Ensembles And The Contributionof A Full-Face Negative Pressure Respirator, Oclla Michele Fletcher

USF Tampa Graduate Theses and Dissertations

Protective clothing ensembles are worn by workers as a barrier to chemical and physical hazards, but can restrict heat loss and increase worker heat stress. The question of whether a respirator adds to heat stress or strain burden is a continuing concern among occupational health professionals. The purpose of this study was to determine if there are differences in heat stress or strain among the current Toxicological Agent Protective (TAP) ensemble and two ensemble variations used in demilitarization of chemical weapons. Four acclimatized adult males wore five ensembles in a balanced design while walking in a climatic chamber at a …

Development And Application Of An F/M Based Anaerobic Digestion Model And The Rt-Ribosyn Molecular Biology Method, Matthew Raymond Cutter

USF Tampa Graduate Theses and Dissertations

A simple anaerobic digestion model has been developed for a continuously-stirred tank reactor (CSTR), which links the specific biogas production rate to the food/microorganism ratio (F/M). The model treats the various microbial populations involved in the sequential biological processes involved in anaerobic digestion as a composite and links the entire biomass specific growth rate directly to the specific biogas production rate. The model was calibrated by determining the specific gas production rate for a range of F/M values using a municipal wastewater seed sludge. The model predictions for steady-state biogas production rates were compared to observed biogas production and volatile …

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

Chemistry and Biochemistry

There is an error in the units of the concentrations of potassium iodide and trehalose described in the experimental details on page 1581. The correct concentrations are 0.6 M potassium iodide and 0.3 M trehalose.

Connections Between Sphingosine Kinase And Phospholipase D In The Abscisic Acid Signaling Pathway In Arabidopsis, Liang Guo, Girish Mishra, Jennifer E. Markham, Maoyin Li, Amanda Tawfall, Ruth Welti, Xuemin Wang

Department of Biochemistry: Faculty Publications

Background: Sphingosine kinase (SPHK) and phospholipaseD(PLD) produce different lipid mediators involved in abscisic acid (ABA) response.

Results: Ablation of SPHKs and PLDα1 attenuates ABA-induced production of LCBPs and PA. Phyto-S1P closes stomata in sphk1, sphk2, but not in pldα1, whereas PA closes stomata in all mutants.

Conclusion: SPHK acts upstream of PLDα1, whereas PLDα1 promotes SPHK.

Significance: The roles of lipid messengers in the ABA signaling pathway are clarified.

Novel Phosducin-Like Protein Binding Partners: Exploring Chaperone And Tumor Suppressor Protein Interactions, Amy Jetaun Gray

Theses and Dissertations

Many proteins cannot fold into their native state without the assistance of one or more molecular chaperones. Chaperonins are an essential class of chaperones that provide an isolated chamber for proteins to fold. CCT, a group II chaperonin found in eukaryotes assists in the folding of actins, tubulins, and many other cellular proteins. PhLP1 is a member of the phosducin protein family that assists CCT in the folding of Gβ and its subsequent assembly with Gγ. However, previous studies have not addressed the scope of PhLP1 and CCT-mediated Gβγ; assembly. The data presented in Chapter 2 shows that PhLP1 plays …

Follicular Dendritic Cells, Human Immunodeficency Virus Type 1, And Alpha 1 Antitrypsin, Xueyuan Zhou

Theses and Dissertations

HIV/AIDS is raging and causing millions of deaths around the world. The major challenge in treating HIV/AIDS is the establishment of HIV reservoirs where the viruse escapes both drug and immune system attempts at eradication. Throughout the course of HIV/AIDS, productive HIV infection occurs primarily in the lymphoid follicles or germinal centers (GC) surrounding follicular dendritic cells (FDC). In the GCs, FDCs trap and maintain infectious HIV for years and provide these infectious viruses to the host cells. FDCs also attract B and T cells into the GCs and increase the ability of CD4+ T cells to be infected. Additionally, …

Optical And Mass Spectrometric Studies Of A Helium Dielectric-Barrier Atmospheric-Pressure Plasma Jet Used As An Ambient Desorption Ionization Source, Matthew Spencer Heywood

Theses and Dissertations

Recently there has been a surge in the field of mass spectrometry centered around the concept of rapid analysis of target analytes with minimal or no sample preparation. The target analyte undergoes desorption from its surface of origin and is subsequently ionized under ambient conditions. The technique is termed ambient desorption/ionization mass spectrometry (ADI-MS). Since the introduction of ADI-MS in 2004, there has been an explosion of research based around the development of novel ambient desorption/ionization (ADI) sources with the capability of desorbing and ionizing a variety of target analytes from various sampling surfaces. One type of ADI source uses …

Development Of A Humanized Hla-A2.1/Dp4 Transgenic Mouse Model And The Use Of This Model To Map Hla-Dp4-Restricted Epitopes Of Hbv Envelope Protein, Zhitao Ru, Wenjun Xiao, Anthony Pajot, Zhihua Kou, Shihui Sun, Benard Maillere, Guangyu Zhao, David M. Ojcius, Yu-Chun Lone, Yusen Zhou

All Dugoni School of Dentistry Faculty Articles

A new homozygous humanized transgenic mouse strain, HLA-A2.1^+/+HLA-DP4^+/+ hCD4^+/+mCD4^−/−IAβ^−/−β2m^−/− (HLA-A2/DP4), was obtained by crossing the previously characterized HLA-A2^+/+β2m−/− (A2) mouse and our previously created HLA-DP4^+/+ hCD4^+/+mCD4^−/−IAβ^−/− (DP4) mouse. We confirmed that the transgenes (HLA-A2, HLA-DP4, hCD4) inherited from the parental A2 and DP4 mice are functional in the HLA-A2/DP4 mice. After immunizing HLA-A2/DP4 mice with a hepatitis B DNA vaccine, hepatitis B virus-specific antibodies, HLA-A2-restricted and HLA-DP4-restricted responses were observed to be similar to those in naturally infected humans. Therefore, the present …

Antifungal Activity Of Lactobacillus Against Microsporum Canis, Microsporum Gypseum And Epidermophyton Floccosum, Jiahui Guo, Brid Brosnan, Ambrose Furey, Elke K. Arendt, Padraigin Murphy

Department of Biological Sciences Publications

A total of 220 lactic acid bacteria isolates were screened for antifungal activity using Aspergillus fumigatus and Aspergillus niger as the target strains. Four Lactobacillus strains exhibited strong inhibitory activity on agar surfaces. All four were also identified as having strong inhibitory activity against the human pathogenic fungi Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. One of the four lactobacilli, namely Lb. reuteri ee1p exhibited the most inhibition against dermatophytes. Cell-free culture supernatants of Lb. reuteri ee1p and of the non-antifungal Lb. reuteri M13 were freeze-dried and used to access and compare antifungal activity in agar plate assays …

Proteins: Form And Function, Roy D. Sleator

Department of Biological Sciences Publications

An overwhelming array of structural variants has evolved from a comparatively small number of protein structural domains; which has in turn facilitated an expanse of functional derivatives. Herein, I review the primary mechanisms which have contributed to the vastness of our existing, and expanding, protein repertoires. Protein function prediction strategies, both sequence and structure based, are also discussed and their associated strengths and weaknesses assessed.

Isotopic Niche Differentiation Among Mammals From A Rainforest In Peninsular Malaysia, Kae Kawanishi, Song Horng Neo Liang, Chris T. Darimont, Thomas E. Reimchen, Melvin E. Sunquist

Biochemistry Collection

We performed stable isotope analysis on eight mammalian species: pig-tailed macaque (Macaca nemestrina), long-tailed macaque (M. fascicularis), dusky leaf monkey (Trachypithecus obscurus), brush-tailed porcupine (Atherurus macrourus), wild boar (Sus scrofa), lesser mouse-deer (Tragulus javanicus), greater mouse-deer (T. napu), and barking deer (Muntiacus muntjac), to test the hypothesis that the differences in diet and habitat types among species, guilds and foraging strategies are reflected in the δ¹⁵N and δ¹³C signatures of their tissues. Whereas the isotopic ratios differed among taxa, the four major isotopic groups observed were: mouse-deer species, primate species, brush-tailed porcupine, and wild boar. The …

Glycoinositolphospholipids From Leishmania Braziliensis And L. Infantum: Modulation Of Innate Immune System And Variations In Carbohydrate Structure, Rafael Ramiro Assis, Izabela Coimbra Ibraim, Fátima Soares Noronha, Salvatore J. Turco, Rodrigo Pedro Soares

Molecular and Cellular Biochemistry Faculty Publications

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important …

Auf1/Hnrnp D Represses Expression Of Vegf In Macrophages, Abigail Fellows, Mary E. Griffin, Brenda L. Petrella, Lihui Zhong, Fatemeh P. Parvin-Nejad, Roy Fava, Peter Morganelli, R. Brooks Robey, Ralph C. Nichols

Dartmouth Scholarship

Vascular endothelial growth factor (VEGF) is a regulator of vascularization in development and is a key growth factor in tissue repair. In disease, VEGF contributes to vascularization of solid tumors and arthritic joints. This study examines the role of the mRNA-binding protein AUF1/heterogeneous nuclear ribonucleoprotein D (AUF1) in VEGF gene expression. We show that overexpression of AUF1 in mouse macrophage-like RAW-264.7 cells suppresses endogenous VEGF protein levels. To study 3′ untranslated region (UTR)–mediated regulation, we introduced the 3′ UTR of VEGF mRNA into a luciferase reporter gene. Coexpression of AUF1 represses VEGF-3′ UTR reporter expression in RAW-264.7 cells and in …

Active Site Mutations Change The Cleavage Specificity Of Neprilysin., Travis Sexton, Lisa J. Hitchcook, David W. Rodgers, Luke H. Bradley, Louis B. Hersh

Molecular and Cellular Biochemistry Faculty Publications

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe⁵⁶³ and Ser⁵⁴⁶. Among the mutants studied in detail we observed changes in their activity towards leucine⁵-enkephalin, insulin B chain, and amyloid β_1-40. For example, NEP^F563I displayed an increase in preference towards cleaving leucine⁵-enkephalin relative to insulin B chain, while mutant NEP^S546E was less discriminating …

Matrix Metalloproteinase Genes Are Transcriptionally Regulated By E2f Transcription Factors: A Link Between Cell Cycle Control And Metastatic Progression, Jacqueline Lea Johnson

USF Tampa Graduate Theses and Dissertations

The RbµE2F transcriptional regulatory pathway plays a critical role in the cell cycle. Rb is inactivated through multiple waves of phosphorylation, mediated mainly by cyclin D and cyclin E associated kinases. Once Rb is inactivated, cells can enter Sµphase. Collectively, three Rb family members and ten E2F proteins coordinate every additional stage of the cell cycle, from quiescence to mitosis. However the RbµE2F pathway is frequently altered in cancer. Aside from cell proliferation, the RbµE2F pathway regulates other essential cellular processes including apoptosis, cell differentiation, angiogenesis and DNA damage repair pathways, but its role in invasion and cancer progression is …

Characterization Of Aquaporin 4 Protein Expression And Localization In Tissues Of The Dogfish (Squalus Acanthias), Christopher P. Cutler, Sheena Harmon, Jonathan Walsh, Kia Burch

Biology: Faculty Publications

The role of aquaporin water channels such as aquaporin 4 (Aqp4) in elasmobranchs such as the dogfish Squalus acanthias is completely unknown. This investigation set out to determine the expression and cellular and sub-cellular localization of Aqp4 protein in dogfish tissues. Two polyclonal antibodies were generated (AQP4/1 and AQP4/2) and these showed somewhat different characteristics in Western blotting and immunohistochemistry. Western blots using the AQP4/1 antibody showed two bands (35.5 and 49.5 kDa) in most tissues in a similar fashion to mammals. Liver had an additional band of 57 kDa and rectal gland two further faint bands of 37.5 and …

Transcriptomic Landscape Of Breast Cancers Through Mrna Sequencing, Jeyanthy Eswaran, Dinesh Cyanam, Prakriti Mudvari, Sirigiri Divijendra Natha Reddy, Suresh Pakala, Sujit S. Nair, Liliana Florea, Suzanne A.W. Fuqua, Sucheta Godbole, Rakesh Kumar

Biochemistry and Molecular Medicine Faculty Publications

No abstract provided.

Dynamics Of The Solvent Exchange Reaction Of Weakly Bound Organic Solvents To Group 6 Transition Metal Carbonyls And The Molybdenum Hexacarbonyl Mediated Pauson-Khand Reaction, Richard J. Gates

Theses and Dissertations

Many organometallic reactions are solvent-dependent, suggesting solvent molecules interact with reaction intermediates. Studies of the solvent exchange reaction of group 6 transition metal carbonyls with moderately binding ligands have provided insight into these interactions, however, studies of the mechanism for this reaction with weakly binding ligands have not been performed. Experiments were conducted on the nanosecond time scale in methylcyclohexane over the temperature range of 4 to 44 °C using Step Scan FTIR (SS FTIR) spectroscopy with weakly binding ligands benzene and mesitylene. Upon photolysis of the metal hexacarbonyls, the kinetically favored product (M(CO)5(solv)), decays following pseudo-first-order kinetics to the …

Down-Regulating Sphingolipid Synthesis Increases Yeast Lifespan, Xinhe Huang, Jun Liu, Robert C. Dickson

Molecular and Cellular Biochemistry Faculty Publications

Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate …

Single-Molecule Analysis Of The Microtubule Cross-Linking Protein Map65-1 Reveals A Molecular Mechanism For Contact-Angle-Dependent Microtubule Bundling, Amanda Tulin, Sheri Mcclerklin, Yue Huang, Ram Dixit

Biology Faculty Research

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also …

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.