Molecular Biology Commons^™

Structure And Dynamics Of Metalloproteins In Live Cells, Jeremy D. Cook, James E. Penner-Hahn, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

X-ray absorption spectroscopy (XAS) has emerged as one of the premier tools for investigating the structure and dynamic properties of metals in cells and in metal containing biomolecules. Utilizing the high flux and broad energy range of X-rays supplied by synchrotron light sources, one can selectively excite core electronic transitions in each metal. Spectroscopic signals from these electronic transitions can be used to dissect the chemical architecture of metals in cells, in cellular components and in biomolecules at varying degrees of structural resolution. With the development of ever-brighter X-ray sources, X-ray methods have grown into applications that can be utilized …

Comparison Of Electrically Mediated And Liposome-Complexed Plasmid Dna Delivery To The Skin, Loree C. Heller, Mark J. Jaroszeski, Domenico Coppola, Richard Heller

Bioelectrics Publications

BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

RESULTS: Experiments …

Molecular Characterisation Of A Bovine-Like Rotavirus Detected From A Giraffe, Emily Mulherin, Jill Bryan, Marijke Beltman, Luke O'Grady, Eugene Pidgeon, Lucie Garon, Andrew Lloyd, John Bainbridge, Helen O'Shea, Paul Whyte, Séamus Fanning

Department of Biological Sciences Publications

Background

Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf.

Results

This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. …

Estimating Kinetic And Thermodynamic Parameters From Single Molecule Enzyme-Inhibitor Interactions, Laura Porter-Peden, Sarah G. Kamper, Mark Vander Wal, Ronald Blankespoor

University Faculty Publications and Creative Works

We report the application of recently developed microscopic models to estimate the apparent kinetic and thermodynamic parameters in a single molecule force spectroscopy study of the carbonic anhydrase enzyme and a complementary sulfonamide inhibitor. The most probable rupture force for the enzyme-inhibitor interaction shows a nonlinear dependency on the log-loading rate. Estimates for the kinetic and thermodynamic parameters were obtained by fitting the nonlinear dependency to linear cubic potential and cusp potential models and compared to the standard Bell-Evans model. The reliability of the estimated parameters was verified by modeling the experimental rupture force distributions by the theoretically predicted distributions …

Dynamics Of Apomyoglobin In The Α-To-Β Transition And Of Partially Unfolded Aggregated Protein, E. Fabiani, A. M. Stadler, D. Madern, M. M. Koza, M. Tehei, M. Hirai, G. Zaccai

Faculty of Science - Papers (Archive)

Changes of molecular dynamics in the α-to-β transition associated with amyloid fibril formation were explored on apo-myoglobin (ApoMb) as a model system. Circular dichroism, neutron and X-ray scattering experiments were performed as a function of temperature on the protein, at different solvent conditions. A significant change in molecular dynamics was observed at the α-to-β transition at about 55 ˚C, indicating a more resilient high temperature β structure phase. A similar effect at approximately the same temperature was observed in holo-myoglobin, associated with partial unfolding and protein aggregation. A study in a wide temperature range between 20 K and 360 K …

Curriculum Vitae, Robert P. Lane

Robert P. Lane

No abstract provided.

Oriented Cell Growth On Self-Assembled Bacteriophage M13 Thin Films, Jianhua Rong, L. Andrew Lee, Kai Li, Brandon Harp, Charlene M. Mello, Zhongwei Niu, Qian Wang

Faculty Publications

Fibrillar M13 bacteriophages were used as basic building blocks to generate thin films with aligned nanogrooves, which, upon chemical grafting with RGD peptides, guide cell alignment and orient the cell outgrowth along defined directions.

The Role Of Gtp Binding And Hydrolysis At The Attoc159 Preprotein Receptor During Protein Import Into Chloroplasts, Danny Schnell, F. Wang, B. Agne, F. Kessler

Danny Schnell

The majority of nucleus-encoded chloroplast proteins are targeted to the organelle by direct binding to two membrane-bound GTPase receptors, Toc34 and Toc159. The GTPase activities of the receptors are implicated in two key import activities, preprotein binding and driving membrane translocation, but their precise functions have not been defined. We use a combination of in vivo and in vitro approaches to study the role of the Toc159 receptor in the import reaction. We show that atToc159-A864R, a receptor with reduced GTPase activity, can fully complement a lethal insertion mutation in the ATTOC159 gene. Surprisingly, the atToc159-A864R receptor increases the rate …

Characterization Of Heat Shock Protein A12b As A Novel Angiogenesis Regulator., Rebecca J. Steagall

Electronic Theses and Dissertations

Previously, we cloned Heat shock protein A12B (HspA12B), the newest member of a recently defined subfamily of proteins distantly related to the Hsp70 family that are enriched in atherosclerotic lesions. We have found that HspA12B is predominantly expressed in vascular endothelium, and that it is involved in angiogenesis which we probed by in vitro angiogenesis assays (Matrigel), migration assays and Directed In Vivo Angiogenesis Assay (DIVAA). Hsp70s are molecular chaperones that are inducible by stress and have been found to be anti-apoptotic (Li et al. 2000; Nylandsted et al. 2000; Garrido et al. 2001). Because of its homology to Hsp70, …

Detection Of Biomarkers By Mrs Requires Error Analysis, Jeffrey C. Hoch, Mark W. Maciejewski, Michael R. Gryk

UCHC Articles - Research

Using MRS imaging and singular value decomposition (SVD), Manganas et al. (Science 318, 980,2007) reported a nuclear magnetic resonance diagnostic for neural progenitor cells. Characteristics of MRS experiments are known to be problematic for SVD-based methods. Absent detection using alternate methods of spectrum analysis or controls to quantify the false discovery rate, little significance can be attached to their result.

Dendrimer Supramolecular Assembly For Gene Delivery, Karthikeyan Pasupathy

All Theses

Dendrimers have found many applications in the fields of polymer science, biophysics, nanomedicine and the petroleum industry. Poly(amidoamine) (PAMAM) was studied as a model dendrimer and squalane as a model hydrocarbon. The interaction between PAMAM and squalane is pH dependent. Specifically, at low or neutral pH the squalane is found on the periphery of the PAMAM while at high pH the hydrocarbon is entrapped inside the PAMAM molecules.

Single-molecule fluorescence revealed that the interaction between PAMAM and squalane is reversible. At a pH value of 8, the time constants for the approaching, binding and dissociation of single PAMAM to squalane …

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

College of the Pacific Faculty Articles

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P_AOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing …

Computational Design Of Orthogonal Ribosomes, Lon Chubiz, Christopher Rao

Biology Department Faculty Works

Orthogonal ribosomes (o-ribosomes), also known as specialized ribosomes, are able to selectively translate mRNA not recognized by host ribosomes. As a result, they are powerful tools for investigating translational regulation and probing ribosome structure. To date, efforts directed towards engineering o-ribosomes have involved random mutagenesisbased approaches. As an alternative, we present here a computational method for rationally designing o-ribosomes in bacteria. Working under the assumption that base-pair interactions between the 16S rRNA and mRNA serve as the primary mode for ribosome binding and translational initiation, the algorithm enumerates all possible extended recognition sequences for 16S rRNA and then chooses those …

Aminoacyl-Trna Synthetase Complexes: Molecular Multitasking Revealed, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. …

In Vivo Murine Melanoma Tumor Responses To Nanosecond Pulsed Electric Field Treatment, Xinhua Chen

Theses and Dissertations in Biomedical Sciences

High intensity nanosecond pulsed electric fields (nsPEF) were applied to melanoma tumors to observe functional and structural biological changes and to investigate the possible molecular mechanisms responsible. An animal model was set up by injecting B16F10 mouse melanoma cells into SKH-1 mice. A treatment (Tx) of 100 pulses: 300 nanosecond duration; 40 kV/cm field strength; at 0.5 Hz rate were delivered to melanoma tumors in 120 mice. The nsPEF Txcaused tumor self-destruction with sharply decreased cell volumes and shrunken nuclei. The apoptotic biochemical tests confirmed nsPEF Tx induced apoptosis in a time-dependent manner. Examination of gross vessel and micro-vessel density …

Pge2 Receptor Subsets Mediate Hcg Regulation Of The Plasminogen Activator/Plasmin System In Primate Periovulatory Follicles, Nune Markosyan

Theses and Dissertations in Biomedical Sciences

Elevated follicular prostaglandin E2 (PGE2) levels are necessary for follicle rupture during ovulation. As the degradation of the follicle wall requires proteolysis, it was hypothesized that regulation of proteolysis by gonadotropins is PGE2 mediated.

There are four PGE2 receptors: EP1, EP2, EP3 and EP4. To determine which PGE2 receptors are present in monkey periovulatory follicles, granulosa cells and whole ovaries were collected 0, 12, 24, and 36 hours after administration of an ovulatory dose of human chorionic gonadotropin (hCG), or 36 hours after hCG from monkeys that received the prostaglandin (PG) synthesis inhibitor celecoxib. All EP receptor mRNAs were present …

Evolution Of Metal(Loid) Binding Sites In Transcriptional Regulators, Efrén Ordóñez, Saravanamuthu Thiyagarajan, Jeremy D. Cook, Timothy L. Stemmler, José A. Gil., Luís M. Mateos, Barry P. Rosen

Biochemistry and Molecular Biology Faculty Publications

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II) and Ni(II). These homodimeric repressors bind to DNA in absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the …

Structural And Functional Mapping Of The Archaeal Multi-Aminoacyl-Trna Synthetase Complex, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Methanothermobacter thermautotrophicus contains a multi-aminoacyl-tRNA synthetase complex (MSC) of LysRS, LeuRS and ProRS. Elongation factor (EF) 1A also associates to the MSC, with LeuRS possibly acting as a core protein. Analysis of the MSC revealed that LysRS and ProRS specifically interact with the idiosyncratic N- and C- termini of LeuRS, respectively. EF-1A instead interacts with the inserted CP1 proofreading domain, consistent with models for post-transfer editing by class I synthetases such as LeuRS. Together with previous genetic data, these findings show that LeuRS plays a central role in mediating interactions within the archaeal MSC by acting as a core scaffolding …

A Cytosolic Iron Chaperone That Delivers Iron To Ferritin, Haifeng Shi, Krisztina Z. Bencze, Timothy L. Stemmler, Caroline C. Philpott

Biochemistry and Molecular Biology Faculty Publications

Ferritins are the main iron storage proteins found in animals, plants and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. The human Poly r(C)-Binding Protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo, and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 …

Writing Research Proposal: Literature Review And Database Search, Mamoudou H. Dicko Prof.

Pr. Mamoudou H. DICKO, PhD

Atf4 Is An Oxidative Stress–Inducible, Prodeath Transcription Factor In Neurons In Vitro And In Vivo, Philipp Lange, Juan Chavez, John T. Pinto, Giovanni Coppola, Chiao-Wang Sun, Tim Townes, Rajiv Ratan

NYMC Faculty Publications

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative …

Structural And Biochemical Investigation Of The Molecular Mechanisms Of Dna Response And Repair In Humans And Escherichia Coli., Steven Michael Shell

Electronic Theses and Dissertations

The genomes of all living cells are under constant attack from both endogenous and exogenous agents that damage DNA. In order to maintain genetic integrity a variety of response pathways have evolved to recognize and eliminate DNA damage. Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is a required factor for all major DNA metabolisms. Although much work has been done to elucidate the nature of the interaction between RPA and ssDNA currently there is no structural information on how the full-length protein binds to ssDNA. This study presents a novel examination of the full nucleoprotein complex …

Collagen 11a1 Is Indirectly Activated By Lymphocyte Enhancer-Binding Factor 1 (Lef1) And Negatively Regulates Osteoblast Maturation, Rachel A. Kahler, Sorcha M.C. Yingst, Luke H. Hoeppner, Eric D. Jensen, David Krawczak, Julia T. Oxford, Jennifer J. Westendorf

Biomolecular Research Center Publications and Presentations

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, characterized by craniofacial abnormalities, nearsightedness and hearing abnormalities. Despite its link to human diseases, few studies have characterized the factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the ß-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted deletion of Col11a1 with an antisense …

The Ttc7Fsn/Fsn Mutation Results In Hyperactivation Of Lymphocytes And Overproduction Of Il-4 Leading To The Development Of Systemic Autoimmunity, Beth Lindroth Hill

Electronic Theses and Dissertations

Ttc7^fsn/fsn mice exhibit systemic autoimmunity characterized by hyperactivated B cells, increased interleukin-4, autoantibodies, kidney disease and reduced lifespan. Because the pathology is similar to systemic lupus erythematosus, Ttc7^fsn/fsn mice are a useful model with which to study early events that lead to autoimmune disease. Although the Ttc7^fsn mutation has been identified the gene function is unknown. The phenotype of Ttc7^fsn/fsn mice mimics the Th2 autoimmunity of the IL-4 transgenic (Erb et al 1997). It was previously unknown whether the over-production of IL-4 was an intrinsic defect of Ttc7^fsn/fsn lymphocytes that led to autoimmunity, or, whether …

Expression Analysis Of Plant Defense Responses During The Establishment Of Biotrophy And Role Of Abiotic Stress In The Infection Of Dyer’S Woad (Isatis Tinctoria) By Puccinia Thlaspeos, Elizabeth Thomas

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The kinetics and amplitude of the salicylic acid-responsive pathogenesis-related (PR) genes and the cytochrome P450 gene ItCYP79B2 in the compatible interaction between Puccinia thlaspeos and dyer’s woad (Isatis tinctoria) during the first 72 hours of inoculation were examined. Immediately following penetration of the host by the rust pathogen, there was a modest up-regulation of PR genes but a significant down-regulation of ItCYP79B2 expression. During haustoria formation, a significant pathogen-mediated suppression of PR genes was observed with a corresponding up-regulation of ItCYP79B2. This potentially facilitates haustoria formation by P. thlaspeos. After haustoria formation, a more …

Lipid Aldehydes Are Substrates And Transcriptional Regulators Of Cytochromes P450., Immaculate Amunom

Electronic Theses and Dissertations

4-hydroxy-2-nonenal (4-HNE) and propene-2-al (acrolein) are highly reactive á,â-unsaturated aldehydes. 4-HNE and acrolein are generated in vivo as products of lipid peroxidation. These aldehydes are implicated in the onset of several diseases including atherosclerosis and neurodegenerative disease. They also react with nucleophilic cellular macromolecules including proteins, DNA and phospholipids. Some of these reactions in vivo lead to inhibition of enzyme activities, depletion of glutathione and oxidative stress. Because of their pathophysiological relevance, attention has focused during the last decade on the ability of mammals to metabolize these lipid aldehydes. The purpose of this study was therefore to determine the role …

Gene Alterations By Peroxisome Proliferator-Activated Receptor Gamma Agonists In Human Colorectal Cancer Cells, Maria Cekanova, X Li, J Yuan, K B. Kim, Seung J. Baek

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear transcription factor that controls the genes involved in metabolism and carcinogenesis. In the present study, we examined the alteration of gene expression in HCT-116 human colorectal cancer cells by PPARgamma agonists: MCC-555 (5 microM), rosiglitazone (5 microM), and 15-deoxy-Delta12,14-prostaglandin J2 (1 microM). The long-oligo microarray data revealed a list of target genes commonly induced (307 genes) and repressed (32 genes) by tested PPARgamma agonists. These genes were analyzed by Onto-Express software and KEGG pathway analysis and revealed that PPARgamma agonists are involved in cell proliferation, focal adhesion, and several signaling pathways. …

Gene Alterations By Peroxisome Proliferator-Activated Receptor Gamma Agonists In Human Colorectal Cancer Cells, Maria Cekanova, J Yuan, X Li, K B. Kim, Seung J. Baek

Maria Cekanova MS, RNDr, PhD

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear transcription factor that controls the genes involved in metabolism and carcinogenesis. In the present study, we examined the alteration of gene expression in HCT-116 human colorectal cancer cells by PPARgamma agonists: MCC-555 (5 microM), rosiglitazone (5 microM), and 15-deoxy-Delta12,14-prostaglandin J2 (1 microM). The long-oligo microarray data revealed a list of target genes commonly induced (307 genes) and repressed (32 genes) by tested PPARgamma agonists. These genes were analyzed by Onto-Express software and KEGG pathway analysis and revealed that PPARgamma agonists are involved in cell proliferation, focal adhesion, and several signaling pathways. …

Treg Depletion Inhibits Efficacy Of Cancer Immunotherapy: Implications For Clinical Trials., James Curtin, Marianela Candolfi, Tamer Fakhouri, Chunyan Liu, Anderson Alden, Matthew Edwards, Pedro Lowenstein, Maria Castro

Articles

BACKGROUND: Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients. FINDINGS: Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs …

Heat Shock Protein 101 Effects In A. Thaliana: Genetic Variation, Fitness And Pleiotropy In Controlled Temperature Conditions., S. J. Tonsor, C. Scott, I. Boumaza, Elizabeth Vierling

Elizabeth Vierling

The Hsp100/ClpB heat shock protein family is ancient and required for high temperature survival, but natural variation in expression and its phenotypic effects is unexplored in plants. In controlled environment experiments, we examined the effects of variation in the Arabidopsis cytosolic AtHsp101 (hereafter Hsp101). Ten wild-collected ecotypes differed in Hsp101 expression responses across a 22 to 40 degrees C gradient. Genotypes from low latitudes expressed the least Hsp101. We tested fitness and pleiotropic consequences of varying Hsp101 expression in 'control' vs. mild thermal stress treatments (15/25 degrees C D/N vs. 15/25 degrees D/N plus 3 h at 35 degrees C …