Biochemistry, Biophysics, and Structural Biology Commons^™

The Rna-Binding Site Of Bacteriophage Qβ Coat Protein, Francis Lim, Marc Spingola, David Peabody

Biology Department Faculty Works

The coat proteins of the RNA bacteriophages Qβ and MS2 are specific RNA binding proteins. Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition. Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qβ. Here we describe the isolation of mutants that define the RNA-binding site of Qβ coat protein, showing that, as with MS2, it resides on the surface of a large β-sheet. Mutations are also described that convert Qβ coat protein to the RNA binding specificity of MS2. …

Distribution Of R-Banded Chromosome Variants In A Bottlenose Dolphin (Tursiops Truncatus) Population, Kristine Anne Gunter

Dissertations and Theses

When combined with observational and behavioral data, chromosome variant analysis provides a means of assessing population structure in bottlenose dolphins, Tursiops truncatus. In this cytogenetic analysis of thirty-six bottlenose dolphins, a distribution of fluorescent R-band variants was catalogued and used as a way to characterize the genetic structure of a Tursiops population inhabiting the Matagorda Bay, Texas, area. Three different population genetic analytical techniques were evaluated for their usefulness in analyzing cytogenetic data. The chromosome variant data obtained from this study was then compared with cytogenetic data gathered from a resident dolphin population residing near Sarasota, Florida.

The purpose …

The Rna-Binding Site Of Bacteriophage Qβ Coat Protein, Francis Lim, Marc Spingola, David S. Peabody

Marc Spingola

Numa Assembles Into An Extensive Filamentous Structure When Expressed In The Cell Cytoplasm, Alejandro Saredi, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects …

Protein-Protein And Protein-Dna Interactions At The Bacteriophage T4 Dna Replication Fork. Characterization Of A Fluorescently Labeled Dna Polymerase Sliding Clamp, Daniel J. Sexton, Theodore E. Carver, Anthony J. Berdis, Stephen J. Benkovic

Chemistry Faculty Publications

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine …

Mechanisms Of Cytokine-Mediated Localized Immunoprotection., Nora Sarvetnick

Journal Articles: Regenerative Medicine

No abstract provided.

The Carboxyl Terminus Of The Bacteriophage T4 Dna Polymerase Is Required For Holoenzyme Complex Formation, Anthony J. Berdis, Patrice Soumillion, Stephen J. Benkovic

Chemistry Faculty Publications

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated ΔC6 exo−, is identical to wild-type exo− polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo− polymerase, the ΔC6 exo− polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo− polymerase was tested as an in vitro inhibitor of …

Circadian Clock-Controlled Genes Isolated From Neurospora Crassa Are Late Night- To Early Morning-Specific, Deborah Bell-Pedersen, Mari L. Shinohara, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs …

Cloning Of Human Acetyl-Coa Carboxylase-Beta And Its Unique Features., Joohun Ha, Jung-Kee Lee, Kyung-Sup Kim, Lee A. Witters, Ki-Han Kim

Dartmouth Scholarship

Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the …

Open-Path Fourier Transform Infrared Studies Of Large-Scale Laboratory Biomass Fires, Robert J. Yokelson, David W. T. Griffith, Darold E. Ward

Chemistry and Biochemistry Faculty Publications

A series of nine large-scale, open fires was conducted in the Intermountain Fire Sciences Laboratory (IFSL) controlled-environment combustion facility. The fuels were pure pine needles or sagebrush or mixed fuels simulating forest-floor, ground fires; crown fires; broadcast burns; and slash pile burns. Mid-infrared spectra of the smoke were recorded throughout each fire by open path Fourier transform infrared (FTIR) spectroscopy at 0.12 cm⁻¹ resolution over a 3 m cross-stack pathlength and analyzed to provide pseudocontinuous, simultaneous concentrations of up to 16 compounds. Simultaneous measurements were made of fuel mass loss, stack gas temperature, and total mass flow up the …

High-Power Broadly Tunable Picosecond Ir Laser System For Use In Nonlinear Spectroscopic Applications, D. E. Gragson, B. M. Mccarty, G. L. Richmond, D. S. Alavi

Chemistry and Biochemistry

We developed a high-power tunable picosecond IR laser system suitable for nonlinear spectroscopic investigations. We employ a Ti:sapphire regenerative amplifier that produces 1.9-ps, 17-cm⁻¹, 800-nm pulses at 1 kHz as a pump source. White-light generation in ethylene glycol and optical parametric amplification in potassium titanyl phosphate are used to produce the IR pulses. The tuning range extends from 2.4 to 3.8 μm in the idler and 1.0 to 1.2 μm in the signal. A total efficiency (signal plus idler) as high as 20% was achieved. The spatial, spectral, and temporal characteristics of the IR beam are presented along …

Pancreatic Expression Of Interleukin-4 Abrogates Insulitis And Autoimmune Diabetes In Nonobese Diabetic (Nod) Mice., Regula Mueller, Troy Krahl, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Diabetes in nonobese diabetic (NOD) mice is a T cell-dependent autoimmune disease. The destructive activities of autoreactive T cells have been shown to be tightly regulated by effector molecules. In particular, T helper (Th) 1 cytokines have been linked to diabetes pathogenesis, whereas Th2 cytokines and the cells that release them have been postulated to be protective from disease. To test this hypothesis, we generated transgenic NOD mice that express interleukin (IL) 4 in their pancreatic beta cells under the control of the human insulin promoter. We found that transgenic NOD-IL-4 mice, both females and males, were completely protected from …

Interleukin-10 Inhibits Tumor Metastasis Through An Nk-Cell Dependent Mechanism, Li-Mou Zheng, David M. Ojcius, F. Garaud, C. Roth, E. Maxwell, Z. Li, H. Rong, J. Chen, X. Y. Wang, J. J. Catino, I. King

All Dugoni School of Dentistry Faculty Articles

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at …

The Effects Of Zinc On Skeletal Alkaline Phosphatase Activity And Skeletal Tissues, Susan L. Hall

Loma Linda University Electronic Theses, Dissertations & Projects

Zinc is essential for normal growth, however, very little is known about the mechanisms by which zinc influences growth. ALP, an ectoenzyme expressed in high levels in osteoblasts, is a zinc-containing metalloenzyme and is important in bone formation. Preliminary studies showed that low levels of zinc could increase ALP specific activity in human osteosarcoma, SaOS-2 cells. Current studies were designed to characterize this zinc effect, test possible mechanisms, and determine whether the effect is physiologically relevant. Incubation with zinc resulted in a time and dose dependent increase in ALP specific activity in the extracts and conditioned media of SaOS-2 cells, …

Oxidized Low Density Lipoprotein And Lysophosphatidylcholine Stimulate Cell Cycle Entry In Vascular Smooth Muscle Cells - Evidence For Release Of Fibroblast Growth Factor-2., Yuh-Cherng Chai, Philip Howe, Paul Dicorletto, Guy Chisolm

Yuh-Cherng Chai

We have previously shown that oxidized low density lipoprotein (LDL) but not native LDL stimulated DNA synthesis in cultured smooth muscle cells (SMC) and that α-tocopherol (vitamin E) inhibited this proliferative response (Lafont, A., Chai, Y. C., Cornhill, J. F., Whitlow, P. L., Howe, P. H., and Chisolm, G. M. (1995) J. Clin. Invest. 95, 1018-1025). The moiety of oxidized LDL that stimulates DNA synthesis and the cellular mechanism for this potentially mitogenic effect are not known. We now report that lipid fractions containing lysophospholipids from oxidized LDL or phospholipase A2-treated native LDL stimulated SMC DNA synthesis as did palmitoyl …

Developmental And Tissue-Specific Expression Of Mouse Pelle-Like Protein Kinase, Marina Trofimova, Amy B. Sprenkle, Melissa Green, Thomas W. Sturgill, Mark G. Goebl, Maureen A. Harrington

Amy Sprenkle

A Truncated Form Of The Pho80 Cyclin Of Saccharomyces Cerevisiae Induces Expression Of A Small Cytosolic Factor Which Inhibits Vacuole Inheritance., Teresa Nicolson, Barbara Conradt, William Wickner

Dartmouth Scholarship

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of …

Application Of Monoclonal Antibodies Against Creatine Kinase Mm And Bb Isoenzymes For Creatine Kinase Mb Isoenzyme Detection, Lei-Chieh Chuang

Chemistry & Biochemistry Theses & Dissertations

Nondiagnostic ECGs and unknown characteristic chest pains increase the diIIiculty of Acute Myocardial Infarction (AMI) diagnosis. Biochemical markers, such as creatine kinase MB isoenzymes (CK-MB) and myoglobin, are employed for the early detection of AMI. Compared to myoglobin, CK-MB reveals a higher specific indicator for AMI detection owing to its abundance in cardiac tissue. Hence, a successful CK-MB detection is expected to improve AMI diagnosis and therapy.

A CK-MB detecting ELISA system was set up based on a sandwich assay principle by using specific CK-B and CK-M monoclonal antibodies. The dimeric nature of the CK-MB isoenzyme provides two epitopes on …

Il-10 Is Necessary And Sufficient For Autoimmune Diabetes In Conjunction With Nod Mhc Homozygosity., Myung-Shik Lee, Regula Mueller, Linda S. Wicker, Laurence B. Peterson, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Contrary to expectations based on in vitro experiments, we previously found that pancreatic IL-10 did not inhibit autoimmune diabetes but accelerated it in an MHC-dependent manner. Therefore, the ability of IL-10 to overcome the absence of all non-MHC diabetes susceptibility (Idd) alleles was studied in transgenic mice expressing pancreatic IL-10 backcrossed to B10.H2g7 congenic mice, which have no Idd alleles other than NOD MHC (H2g7). IL-10 transgenic backcross 1 (BC1) mice with H2g7/g7 haplotype developed clear-cut insulitis and diabetes, but neither transgenic mice with the H2g/b haplotype nor nontransgenic BC1 mice did so. Further implicating IL-10 in autoimmune diabetes, anti-IL-10 …

Selenocysteine, Identified As The Penultimate C-Terminal Residue In Human T-Cell Thioredoxin Reductase, Corresponds To Tga In The Human Placental Gene, Vadim Gladyshev, Kuan-Teh Jeang, Thressa C. Stadtman

Vadim Gladyshev Publications

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenuim metabolism in human T cells. One of the major ⁷⁵Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were …

Characterization Of Vasoactive Intestinal Peptide Receptors On Human Megakaryocytes And Platelets, Sang K. Park, Thomas A. Olson, Nuran Ercal, Monica Summers, M. Sue O'Dorisio

Chemistry Faculty Research & Creative Works

Vasoactive intestinal peptide receptor I (VIPRI) expression was examined in megakaryocytes using reverse transcriptase-polymerase chain reaction (RT- PCR). VIPRI protein was characterized in platelet membranes using covalent crosslinking techniques. Human megakaryocytes were isolated from suspension cultures of cord blood and adult bone marrow mononuclear cells using a murine monoclonal antibody to human platelet glycoprotein IIB/IIIA (CD41) and immunomagnetic beads. RT-PCR primers were constructed for the VIP, VIPRI, and VIPRII genes as well as for megakaryocyte specific genes, c-mpl and platelet factor 4 (PF-4). VIP, VIPRI, c-mpl, and PF-4 were coexpressed in megakaryocyte mRNA. Southern blot analysis confirmed the expression of …

Anion Induced Blue To Purple Transition In Bacteriorhodopsin, Mrunalini Pattarkine, Anil K. Singh

Faculty Works

Purple membrane (PM, λ" role="presentation">λmax" role="presentation">max 570 nm) of H. halobium on treatment with sulphuric acid changes its colour to blue (λ" role="presentation">λmax" role="presentation">max 608 nm). The purple chromophore can be regenerated from the blue chromophore by exogeneous addition of anions such as CI−" role="presentation">− and HPO42−" role="presentation">2−4. Chloride ion is found to be more effective than the dibasic phosphate ion in regenerating the purple chromophore. Nevertheless, one thing common to the anion regeneration is that both CI−" role="presentation">− and HPO42−" role="presentation">2−4 show marked pH effect. At pH 1.0 the efficiency of …

Progesterone Regulation Of Insulin Like-Growth Factor : Binding Protein-5 Gene Transcription In Human Osteoblasts, Viroj Boonyaratanakornicit

Loma Linda University Electronic Theses, Dissertations & Projects

Insulin-like growth factor binding protein-5 (IGFBP-5) is produced by osteoblasts and potentiates IGF mitogenic actions in osteoblast cultures. Progesterone (PG) increases osteoblast proliferation and increases osteoblast IGFBP-5 expression. Mechanisms underlying PG induction of IGFI3P-5 expression were investigated in U2 human osteosarcoma cells. In nuclear run-on analyses, PG increased IGF)3P-5 gene transcription to 400% of control. To define the underlying mechanisms, the human IGF13P-5 gene proximal promoter was cloned and sequenced. The proximal 500 bp of this region contains TATA and CAAT boxes, five putative PG response element (PRE) half-sites and two tandem CACCC boxes. Chloramphenicol acetyltransferase (CAT) reporter constructs containing …

Ultrasound Three- Dimensional Velocity Measurements By Feature Tracking, Gregory R. Bashford, Olaf T. Von Ramm

Biomedical Imaging and Biosignal Analysis Laboratory

This article describes a new angle-independent method suitable for three-dimensional (3-D) blood flow velocity measurement that tracks features of the ultrasonic speckle produced by a pulse echo system. In this method, a feature is identified and followed over time to detect motion. Other blood flow velocity measurement methods typically estimate velocity using one- (1-D) or two-dimensional (2-D) spatial and time information. Speckle decorrelation due to motion in the elevation dimension may hinder this estimate of the true 3-D blood flow velocity vector. Feature tracking is a 3-D method with the ability to measure the true blood velocity vector rather than …

A Novel Iron-Regulated Metal Transporter From Plants Identified By Functional Expression In Yeast., David Eide, Margaret Broderius, Jeanette Fett, Mary Lou Guerinot

Dartmouth Scholarship

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, …

Phylogenetic Studies Of The United States Bluetongue Viruses And Characterization Of The Viral Vp4 Protein, I-Jen Huang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4.

The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region …

Structure Of A Bovine Thrombin-Hirudin51-65 Complex Determined By A Combination Of Molecular Replacement And Graphics. Incorporation Of Known Structural Information In Molecular Replacement, Jacqueline Vitali, Philip D. Martin, Michael G. Malkowski, Cris M. Olsen, Paul H. Johnson, Brian F.P. Edwards

Physics Faculty Publications

Crystals of the bovine thrombin-hirudins_51-65 complex have space group P6₁22 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in …

Critical Pressures In Multicomponent Lipid Monolayers, John P. Hagen, Harden M. Mcconnell

Chemistry and Biochemistry

Epifluorescence microscopy has been used previously to study coexisting liquid phases in lipid monolayers of dihydrocholesterol and dimyristoylphosphatidylcholine at the air/water interface. This binary mixture has a critical point at room temperature (22°C), a monolayer pressure of approx. 10 mN/m, and a composition in the vicinity of 20-30 mol% dihydrocholesterol. It is reported here that this critical pressure can be lowered, raised, or maintained constant by systematically replacing molecules of this phosphatidylcholine with molecules of a phosphatidylethanolamine, or an unsaturated phosphatidylcholine, or mixtures of the two, while maintaining the dihydrocholesterol concentration at 20 mol%. Thus, even complex mixtures of lipids …

Report Of Significant Findings--Las Vegas Bay/Boulder Basin Investigations, James F. Labounty, Michael Horn, Bureau Of Reclamation

Publications (WR)

Field sampling was carried out between 0830 and 1500 beginning at the confluence of Las Vegas Wash and the Inner Las Vegas Bay. Ten (10) locations were sampled, each in a similar manner. Locations of sampling stations are in line from the Wash-Bay confluence to a point midway between Saddle and Black Islands. In addition, sampling was done at a location midway between Sentinel Island and the base of Fortification Hill, and at the buoy line in front of Hoover Dam. Sampling stations are labeled from LV01, at Wash-Bay confluence, to LV17 at Hoover Dam. A significant data collection point …

Characterization Of Crystalline Formate Dehydrogenase H From Escherichia Coli, Vadim N. Gladyshev, Jeffrey C. Boyington, Sergei V. Khangulov, David Grahame, Thressa C. Stadtman, Peter D. Sun

Vadim Gladyshev Publications

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenumcoordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 μg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations ( 3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 Å resolution …