Biochemistry, Biophysics, and Structural Biology Commons^™

Dissecting The Biosyntheses Of Gilvocarcins And Ravidomycins, Madan Kumar Kharel

University of Kentucky Doctoral Dissertations

Gilvocarcin V (GV) and ravidomycin (RMV) exhibit excellent antitumor activities in the presence of near-UV light at low concentration maintaining a low in vivo cytotoxicity. Although, the exact molecular mechanism for in vivo actions of these antibiotics has yet to be determined, a [2+2] cycloaddition reaction of the vinyl side chain with DNA thymidine residues in addition to the inhibition of topoisomerase II and DNAhistone H3 cross-linking are reported for the GV’s mechanism of action. Such activities have made these molecules interesting candidates for the biosynthetic investigation to generate analogues with improved activity/solubility. Previous biosynthetic studies have suggested that the …

The Globin Gene Family Of The Cephalochordate Amphioxus: Implications For Chordate Globin Evolution, Bettina Ebner, Georgia Panopoulou, Serge N. Vinogradov, Laurent Kiger, Michael C. Marden, Thorsten Burmester, Thomas Hankeln

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire.

Results

We show that B. floridae harbors at least fifteen paralogous globin genes, …

Stream Invertebrate Responses To A Catastrophic Decline In Consumer Diversity, Jose Checo Colón-Gaud, Matt R. Whiles, Karen R. Lips, Catherine M. Pringle, Susan Kilham, Roberto Brenes, Scot D. Peterson

Biology: Faculty Publications

Tadpoles are often abundant and diverse consumers in headwater streams in the Neotropics. However, their populations are declining catastrophically in many regions, in part because of a chytrid fungal pathogen. These declines are occurring along a moving disease front in Central America and offer the rare opportunity to quantify the consequences of a sudden, dramatic decline in consumer diversity in a natural system. As part of the Tropical Amphibian Declines in Streams (TADS) project, we examined stream macroinvertebrate assemblage structure and production for 2 y in 4 stream reaches at 2 sites in Panama. One site initially had healthy amphibians …

Cell-Cell Junction Signaling Regulating Dna Double-Strand Break Repair In Breast Cells, Sinduja Ethiraj

Theses and Dissertations

Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that …

Automated Interpretation Of The Tandem Mass Spectra Of Peptides Using Artificial Neural Networks, Timothy Patrick Manning

Theses

The manual interpretation of mass spectra is a complex and time consuming task. The problem of manually interpreting this data is further exacerbated by the large numbers of mass spectra which can potentially be produced in a single proteoniics experiment. This shows the need for high throughput approaches to the interpretation of mass spectra. Existing automated approaches are however error prone due to the complexity of the task. Accordingly, this thesis discusses and evaluates the application of neural networks to improving the sensitivity, specificity and robustness of current approaches to the automated interpretation of such mass spectral data.

Several neural …

Purification And Properties Of Amycolatopsis Mediterranei Dsm 43304 Lipase And Its Potential In Flavour Ester Synthesis, Dharmendra Dheeman, Gary Henehan, Jesus Maria Frias

Articles

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in 398 fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) under optimal temperature (60°C) and pH (8.0) conditions were 0.10 ± 0.01 mM and 2.53 ± 0.06 mmol/minmg, respectively. The purified …

Improving Predicted Protein Loop Structure Ranking Using A Pareto-Optimality Consensus Method, Yaohang Li, Ionel Rata, See-Wing Chiu, Erik Jakobsson

Computer Science Faculty Publications

Background

Accurate protein loop structure models are important to understand functions of many proteins. Identifying the native or near-native models by distinguishing them from the misfolded ones is a critical step in protein loop structure prediction.

Results

We have developed a Pareto Optimal Consensus (POC) method, which is a consensus model ranking approach to integrate multiple knowledge- or physics-based scoring functions. The procedure of identifying the models of best quality in a model set includes: 1) identifying the models at the Pareto optimal front with respect to a set of scoring functions, and 2) ranking them based on the fuzzy …

Induction Of Interferon And Interferon Signaling Pathways By Replication Of Defective Interfering Particle Rna In Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins, Debasis Panda, Phat X. Dinh, Lalit Beura, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN- promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome …

Towards An Understanding Of The Herpes Simplex Virus Type 1 Latency-Reactivation Cycle, Guey-Chuen Perng, Clinton Jones

School of Veterinary and Biomedical Sciences: Faculty Publications

Infection by herpes simplex virus type 1 (HSV-1) can cause clinical symptoms in the peripheral and central nervous system. Recurrent ocular shedding can lead to corneal scarring and vision loss making HSV-1 a leading cause of corneal blindness due to an infectious agent. The primary site of HSV-1 latency is sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs resulting in virus transmission and recurrent disease. During latency, the latency-associated transcript (LAT) is abundantly expressed. LAT expression is important for the latency-reactivation cycle in animal models, in part, because it inhibits apoptosis, viral gene expression, and productive infection. A …

Amino Acid Transport In Thermophiles: Characterization Of An Arginine-Binding Protein In Thermotoga Maritima, Matthew S. Luchansky, Bryan S. Der, Sabato D'Auria, Gabriella Pocsfalvi, Luisa Iozzion, Daniela Marasco, Jonathan D. Dattelbaum

Chemistry Faculty Publications

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 °C. Its multimeric structure may provide further evidence of …

Amino Acid Transport In Thermophiles: Characterization Of An Arginine-Binding Protein In Thermotoga Maritima. 2. Molecular Organization And Structural Stability, Andrea Scirè, Anna Marabotti, Maria Staiano, Luisa Iozzion, Matthew S. Luchansky, Bryan S. Der, Jonathan D. Dattelbaum, Fabio Tanfani, Sabato D'Auria

Chemistry Faculty Publications

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine …

Plasticity Of Acquired Secondary Metabolites In Clathria Prolifera (Demospongia: Poecilosclerida): Putative Photoprotective Role Of Carotenoids In A Temperate Intertidal Sponge, Jonathan D. Dattelbaum, Drew Sieg, Malcolm Hill, Chris M. Manieri, Giles Thomson

Chemistry Faculty Publications

Several marine sponges sequester high concentrations of carotenoids in their tissues. The diversity of carotenoid compounds has been described in detail for a handful of species, but to date, little attention has been paid to natural variability in the concentration and constituency of carotenoid pools. Also lacking are experimental tests of some of the proposed adaptive benefits of carotenoids to the sponge. To address some of these deficits in our understanding of sponge ecology, we used a combination of analytic chemistry, field surveys, and manipulative experiments to determine what function these compounds might play. Attention was focused on the common, …

Ipla2Β, Alternative Splicing And Apoptosis Of Pancreatic Islets, Bhargavi Emani

Theses and Dissertations

Ceramides are bioactive lipids that can promote splicing of apoptosis-related genes, including caspase 9 and BCL-x. A recent study demonstrated that expression of neutral sphingomyelinase (NSMase), an enzyme that hydrolyzes sphingomyelins to generate ceramide, is regulated by Group VIA phospholipase A2 (iPLA2β)-dependent mechanism during β-cell apoptosis. This prompted us to hypothesize that iPLA2 is upstream of ceramide generation in the process regulating splicing of apoptotic genes. To test this, Jurkat T cells were treated with the selective inhibitor of iPLA2β, bromoenol lactone (BEL), RNA was isolated and converted to cDNA, and caspase 9 and BCL-x mRNA viii species were amplified …

Isolation, Characterization And Molecular Cloning Of Dnase Iiib From Drosophila Melanogaster, Brenda Cristina Anchondo

Open Access Theses & Dissertations

Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββα-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA non-specific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar …

Evaluating The Role Of Evolutionarily Conserved Regions Of Ledgf/P75 In Hiv-1 Infection, Jose Adrian Garcia

Open Access Theses & Dissertations

LEDGF/p75 is an important cellular co-factor for lentiviral integration. LEDGF/p75-deficient cells are markedly resistant to HIV-1 infection and re-expression of the wild type protein rescues infectivity. Although the molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet known, this co-factor activity requires the interaction of LEDGF/p75 with both the host chromatin and viral integrase. In order to evaluate the involvement of other LEDGF/p75 regions in HIV-1 infection we constructed a panel of deletion mutants targeting clusters of charged residues that are evolutionarily conserved and predicted to be post-translationally modified. These mutants were evaluated for their ability to rescue HIV-1 …

Interactions Of The Cellular Sumoylation System With Influenza A Virus And Its Non-Structural Protein Ns1a (Ns1a), Sangita Pal

Open Access Theses & Dissertations

The most important current anti-influenza weapons, vaccination and antiviral drugs, can be rapidly rendered fully ineffective thanks to the virus's high mutational rate, which produces viruses exhibiting new antigenic properties and structural proteins insensitive to the drug's mechanism of action. One attractive alternative is to develop drugs that modulate the activity of cellular systems either required for viral growth or able to neutralize viral growth. Here we demonstrate that the cellular SUMOylation system, a post-translational modification involving the conjugation of the Small Ubiquitin-like MOdifier (SUMO) to specific protein targets using a Ubiquitin-like enzymatic cascade, interacts closely with influenza virus during …

A Specific Regulatory Role For Sgtα On The Maturation And Activation Of Steroid Hormone Receptors, Atanu Paul

Open Access Theses & Dissertations

Steroid hormone receptors (SHRs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. These receptors regulate various physiological functions in higher ordered eukaryotes. In the absence of hormone these receptors form a complex with molecular chaperones such as Hsp90 and Hsp70 and other cochaperones in the cytoplasm. Association with the Hsp90-Hsp70 chaperone machinery leads to the maturation of the Ligand Binding Domain (LBD) of the receptors and enables the receptors to bind hormone with high affinity. Upon binding with a specific hormone the receptors are translocated into the nucleus where they initiate transcription of specific genes. Although the involvement …

Computer Aided Drug Design Methods & Quantitative Structure-Activity/Property Relationships, Suman Sirimulla

Open Access Theses & Dissertations

The first part of dissertation consists of development of a QSAR model for 229 mutagenic aromatic amines and a QSPR model of partial molar volumes of amino acids. A common procedure for QSAR analysis consist of data selection (generally sets of homologous series of compounds and their corresponding biological activities), tabulation of trial physicochemical or molecular structural descriptors, followed by a multilinear statistical analysis to derive a statistically valid QSAR correlation of the activity data making use of a subset of the trial descriptors. A final important step is cross-validation to assess the putative predictive (rather than just correlative) capabilities …

Biochemical Characterization Of Suppressor Of Ikk-Ε And Nak-Associated Protein 1, Jonathan Forbes

Theses and Dissertations

Innate immunity provides the first line of defense against invading pathogen by recognizing and mounting a response to the pathogenic challenge. Among the cellular mechanisms of the innate immune response, Toll-like receptor 3 (TLR3) recognizes viral dsRNA and signals subsequent production of type-I interferon. The TLR3:interferon signaling cascades contains a kinase complex composed of two kinases and a scaffold protein, NAK-associated protein 1 (NAP1). The role of NAP1 in modulating kinase activation or regulation is unknown. A key inhibitory protein identified in the TLR3:interferon pathway, silencer of inhibitor of κBα kinase ε (SIKE), blocks the activity of this kinase complex …

Influence Of Retinal States On The Development And Maintenance Of Retinofugal Projections, Duncan Morhardt

Theses and Dissertations

Vision provides a critical interface with the physical world. This work examines visual development and vision loss in mice to glean the influence of the retinal state on visual connections. I first assessed the impact of retinal activity on the eye-specific segregation of retinal afferents in the lateral geniculate nucleus (LGN) of young Gβ5 -/- mice. Gβ5 is the fifth member of the β subfamily of heterotrimeric G proteins. Gβ5 binds and stabilizes the R7 family of regulators of G-protein signaling (RGS), which accelerate Gi/o GTP hydrolysis. Gβ5 -/- mice, which lack R7RGS activity, have malformed synapses in the outer …

Prospects And Pits On The Path Of Biomimetics: The Case Of Tooth Enamel, Vuk Uskoković

Pharmacy Faculty Articles and Research

This review presents a discourse on challenges in understanding and imitating the process of amelogenesis in vitro on the molecular scale. In light of the analysis of imitation of the growth of dental enamel, it also impends on the prospects and potential drawbacks of the biomimetic approach in general. As the formation of enamel proceeds with the protein matrix guiding the crystal growth, while at the same time conducting its own degradation and removal, it is argued that three aspects of amelogenesis need to be induced in parallel: a) crystal growth; b) protein assembly; c) proteolytic degradation. A particular emphasis …