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A Method For The Large Scale Purification Of Hcg (Human Chorionic Gonadotropin) From The Urine Of Pregnant Women, Chun-Shyan Kuo

Chemistry & Biochemistry Theses & Dissertations

A large scale preparation of human chorionic gonadotropin (HCG) from the urine of pregnant women was accomplished by using kaolin adsorption, NH₄OH extraction and DEAE-cellulose ion exchange. A recovery about 81% of HCG and a specific activity in the 2,000-3,000 IU/mg range was achieved using this purification method.

Two HCG activity fractions, HCG I & HCG II, were isolated from DEAE-cellulose chromatography by a stepwise sodium chloride gradient elution. HCG I was eluted at 0.01-0.02 M NaCl while HCG II was eluted at 0.12-0.02 M NaCl.

The differences between HCG I and HCG II have been studied. The …

The Characterization Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Vicky L. Montoya, Wendy L. Murdoch, Roy C. Ogle, John L. Keating, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Faculty Publications

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they …

Modulation Of Queuine Uptake In Cultured Human Fibroblasts By Phorbol Esters And Interferons, Debra L. Crane

Chemistry & Biochemistry Theses & Dissertations

Queuosine, a nucleoside found exclusively in the first position of the anticodon of transfer RNA (tRNA), is generated post-transcriptionally in an exchange of guanine for queuine by the modifying enzyme tRNA-guanine ribosyltransferase. Phorbol ester tumor promoters were shown to induce queuine hypomodified tRNA, and that phorbol ester action was due, in part, to inhibition of queuine transport across the cell membrane. An unidentified protein designated conditioned medium factor (CMF) that potentiated a phorbol-induced inhibition of queuine uptake was also documented. We suggest here that phorbol ester-induced inhibition of queuine uptake is not a significant factor in inducing queuine-deficient tRNA although …

Molecular Study Of The B19 (Human) Pathogenic Parvovirus, Jamshed Ayub

Theses and Dissertations in Biomedical Sciences

The B19 (human) parvovirus is a small single stranded DNA virus of 5.4 kilobases. B19 is specific for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 differs from other parvoviruses in some important aspects, which include the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter. B19 has an unusual transcription map which is described in this study.

The transcription map of …

Laminin Receptors For Neurite Formation, H. K. Kleinman, Roy C. Ogle, F. B. Cannon, C. D. Little, T. M. Sweeney, L. Luckenbill-Edds

School of Medical Diagnostics & Translational Sciences Faculty Publications

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein …

Incorporation Of A Product Of Mevalonic Acid Metabolism Into Proteins Of Chinese Hamster Ovary Nuclei, Michael Sinensky, L. A. Beck, T. Hosick

Faculty Publications, Biological Sciences

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. …

Comparison Of Cyclic Nucloetide Phosphodiesterase In Physarum Favicomum, C. Kay Holtman, Tammy K. Ebsen, Judith A. Bean, J. Scott Bryles, Thomas J. Lynch

Journal of the Arkansas Academy of Science

We have studied both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase in the myxomycete Physarum flavicomum. The cyclic AMP phosphodiesterase preparations were isolated from both the diploid plasmodial stage of the lifecycle and the haploid myxamoebal stage. The plasmodial enzyme was prepared from spent medium (extracellular) and also from purified nuclei. The myxamoebal enzyme was prepared from purified nuclei. Cyclic GMP phosphodiesterase activity was studied in purified nuclei isolated from the plasmodium. One unusual feature of all the enzymes from the plasmodium is extreme heat stability; they remain catalytically active even after exposure to a boiling water bath for twenty …

The Purification Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Roy C. Ogle, John L. Keating, Vicky L. Montoya, Wendy L. Murdoch, Robert M. Grainger

School of Medical Diagnostics & Translational Sciences Faculty Publications

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then …

Incorporation Of A Product Of Mevalonic Acid Metabolism Into Proteins Of Chinese Hamster Ovary Nuclei, Michael Sinensky, L. A. Beck, T. Hosick

Michael Sinensky

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. …

The Refined Crystal Structure Of A Fully Active Semisynthetic Ribonuclease At 1.8 Å Resolution, Philip D. Martin, Marilynn S. Doscher, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the …

Short-Term Feedback Regulation Of Camp By Accelerated Degradation In Rat Tissues, Tom W. Gettys, Peter F. Blackmore, J. Bruce Redmon, Stephen J. Beebe, Jackie D. Corbin

Bioelectrics Publications

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes. The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pClΦS-cAMP (where 8-pClΦS- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were …

Photoreactivation Of Lethal Damage Induced In Hamster X Xenopus Hybrid Cells And Their Parentals By Uv Light, David Bohlender, Stuart Williams, Emir Cruz, H. Gaston Griggs

Journal of the Arkansas Academy of Science

A85 Xenopus cells that exhibited a high level of photoreactivation (PR) and V79B2 hamster cells that exhibited little PR were fused to produce the V79B2 x A85 cell line — a hybrid line which possessed a relatively stable karyotype, with most cells containing the entire V79B2 and A85 genomes. UV and UV plus PR fluence-survival relations were then determined and compared for the hybrid and parental lines in a first attempt to elucidate interactions of the parental PR mechanisms in the hybrid. It was anticipated that the A85 genome in the hybrid would produce PR enzyme in sufficient concentration and …

Biochemical And Histological Evaluation Of Normal Human Epidermis Vs Cultured Human Epidermis, Ngo Thi Bich Lien

Biological Sciences Theses & Dissertations

Cultured human keratinocytes were grown in vitro for transplantation purposes in case of severe burns. Single keratinocytes were harvested from human epidermis and grown in culture flasks such that multilayered sheets of keratinocyte cells were harvestable in approximately three weeks. In order to evaluate cultured epidermis for equivalency with normal human epidermis, biochemical and histological studies were performed. Thin sections of cultured epidermis were shown to be composed of cells that had undergone developmental differentiation, but not terminal differentiation. Biochemical analyses of keratin and prekeratin proteins isolated from cultured epidermis indicated that cultured keratinocytes have electrophoretic patterns similar to keratins/prekeratins …

Yeast Mannans: Use As A Transport Vehicle For Peptides, Jacqueline Cesario Lee

Masters Theses

Yeast mannans are phosphorylated glycopeptides found in various species of yeast. Their similarity to the mannose-phosphate signal for intracellular lysosomal uptake led mannans to be considered as a possible vehicle for increasing cellular affinity for peptides. Insulin was used as a model peptide and was conjugated to mannans by two coupling methods. The conjugated mannan-insulins were compared to insulin on the basis of cellular binding and ability to induce typical insulin biological effects.

Optimal Conditions For Kinetic Study Of Succinate Dehydrogenase In Rat Liver, Collie B. Shaw, Tara L. Chronister, John D. Peck

Journal of the Arkansas Academy of Science

Succinate dehydrogenase (SDH) commonly is assayed as a marker enzyme for mitochondrial activity. The literature presents numerous conditions for conducting this assay due to the fact that, it has been difficult to get sufficient reduction of the acceptor dye, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). This study was undertaken to optimize the SDH-catalyzed reduction of TTC dye by evaluation of a greater range of molor ratios of TTC to succinate and by further evaluation of additives reported as beneficial. Improvement in enzyme specific activity was achieved by liver perfusion via the left cardiac ventricle with homogenizing solution. Increase in TTC from 1 to …

Effects Of Juglone (5'-Hydroxy-1, 4-Naphthoquinone) On The Algae Anabaena Flos-Aquae, Nostoc Commune, And Scenedesmus Acuminatus, V. Diane Randall, Jimmy D. Bragg

Journal of the Arkansas Academy of Science

Three species of algae, Anabaena flos-aquae, Nostoc commune and Scenedesmus acuminatus were selected for their sensitivity to juglone and studied for the effects of juglone concentrations of 10, 1, 0.5, 0.1 and 0.01 μg/ml upon their growth. A. flos-aquae was most sensitive, with significant inhibition by the 0.5 μg/ml concentration. N. commune was inhibited least, with significant inhibition only in the 10 μg/ml concentration. S. acuminatus was found to be moderately inhibited at the 0.5 μg/ml concentration. All species were found to be non-viable after 14 days exposure to 10 μg/ml juglone. Tests with 7 and 14 day old cells …

Isolation Of Nuclei From Physarum Flavicomum: Demonstration Of Nuclear Cyclic Acid Amp Phosphodiesterase, Judith A. Bean, Maurice G. Kleve, Thomas J. Lynch

Journal of the Arkansas Academy of Science

Cyclic AMP phosphodiesterase activity in the nucleus of the myxomycete Physarum flavicomum was demonstrated by cytochemical staining utilizing electron microscopy and by enzymatic assays with tritiated cyclic AMP as the substrate. Cytochemical staining showed Physarum's plasmodial phosphodiesterase activity to be located in the nucleus, along the plasma membrane, in vesicles, and free in the cytoplasm. Nuclear phosphodiesterase, which may be cell cycle dependent, was primarily located in the nucleolus. Nuclei from three to five day old microplasmodial cultures were isolated by the method of Henney and Yee. Whole cells were collected through centrifugation and washed. Pellets were homogenized in a …

Solid Phase Extraction Of Mammalian Cell Mitochondrial Dna And Its Electrophoretic Separation In Agarose Gels, Theresa Brick-Miller

Biological Sciences Theses & Dissertations

An improved and efficient electrophoretic procedure for the isolation of mitochondrial DNA (mtDNA) is described. The solid phase extraction procedure for the isolation of mtDNA involves embedding as few as 200,000 Ehrlich ascites tumor cells into a block of agarose, digestion of cellular membranes by detergent action and subsequent electrophoretic separation of nucleic acids in agarose gels. Genomic DNA remains in the original block of agarose while the mtDNA migrates in the separation gel to a position equivalent to that obtained for mtDNA isolated by traditional procedures.

The efficacy of a large number of detergents, agarose concentrations, embedding conditions, embedding …

Isolation And Characterization Of Temperature-Sensitive Protein Synthesis Mutants Of Escherichia Coli By Directed Mutagenesis Of The Defective Bacteriophage Lambda Fus2, Kenton L. Lohman

Electronic Theses and Dissertations

Mutagenesis of the defective transducing bacteriophage lambda fus2 was used to isolate a collection of temperature-sensitive mutants of E. coli in the major ribosomal protein gene cluster. Four mutants were examined in detail. Two of the mutants were resistant to the ribosomal antibiotics neamine and spectinomycin. Another mutant was defective in 50S ribosomal subunit assembly at 42(DEGREES)C. The 30S subunit proteins S17 and S19 were changed in two different mutants. Each protein migrated as a more basic species in two-dimensional gels of ribosomal proteins. Ribosomes from each of the four mutants examined showed a temperature-dependent reduction in translational activity in …

Discriminative Insulin Antagonism Of Stimulatory Effects Of Various Camp Analogs On Adipocyte Lipolysis And Hepatocyte Glycogenolysis, Stephen J. Beebe, J. Bruce Redmon, Peter F. Blackmore, Jackie D. Corbin

Bioelectrics Publications

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity …

Lac Operon Expression In Steady State Cells Of Escherichia Coli, Sungae Cho

Dissertations, Theses, and Masters Projects

No abstract provided.

Camp-Dependent Protein Kinase Activation Lowers Hepatocyte Camp, Jackie D. Corbin, Stephen J. Beebe, Peter F. Blackmore

Bioelectrics Publications

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent …

Characterization Of Kpni Interspersed, Repetitive Dna Sequence Families And Their Association With The Nuclear Matrix, Joseph A. Chimera

Electronic Theses and Dissertations

The KpnI, 1.2 and 1.5 kb families of interspersed repetitive DNAs from the African green monkey genome were isolated and characterized. Each family contains three populations of segments based on their sequence lengths and susceptibility to cleavage by the restriction enzymes KpnI and RsaI. The first population contains the smallest segments which are susceptible to both KpnI and RsaI cleavage and have fragment lengths of 1.2 kb (1.2 kb family) and 1.5 kb (1.5 kb family) respectively. The members in this population are referred to as KpnI-sensitive segments. The second population contains longer segments (> 2 kb) which represent fusions …

Purification And Characterization Of A Camp- And Ca2+-Calmodulin-Independent Glycogen Synthase Kinase From Porcine Renal Cortex, Stephen J. Beebe, Erwin M. Reimann, Keith K. Schlender

Bioelectrics Publications

We recently reported the partial purification of a cAMP-independent and Ca²⁺-calmodulin-independent glycogen synthase kinase from porcine renal cortex (Schlender, K. K., Beebe, S. J., and Reimann, E. M. (1981) Cold Spring Harbor Conf. Cell Proliferation, 389-400). Subsequent purification indicated that the enzyme preparation consisted of at least three forms of glycogen synthase kinase which could be resolved by ATP gradient elution from aminoethylphosphate-agarose (AEP-agarose). The predominant form of glycogen synthase kinase, which eluted from AEP-agarose between 2 and 6 mM ATP, was purified approximately 800-fold and is designated GSK-A1. It had a molecular weight of 45,000-50,000 as determined …

Extracellular Phosphodiesterase From The Growth Medium Of The Myxomycete Physarum Flavicomum, Thomas J. Lynch, Mary E. Farrell

Journal of the Arkansas Academy of Science

The plasmodium of the myxomycete Physarum flavicomum secretes cyclic AMP phosphodiesterase into the medium. The extracellular enzyme had a pH optimum between 7 and 8 and a Km of about 500 μM cyclic AMP and was inhibited by theophylline, caffeine and 3-isobutyl-l-methyxanthine (MIX). A marked decrease of enzyme activity was noted in the presence of EDTA, suggesting the requirement of Mg+ + by the enzyme. Addition of Mg+ + and Ca + + stimulated the enzyme while Zn+ + , Co+ + , Pb+ + , Mn+ + , Fe + + + , Ni+ + , and Cu + …

Microheterogeneity Of Type Ii Camp-Dependent Protein Kinase In Various Mammalian Species And Tissues, Alison M. Robinson-Steiner, Stephen J. Beebe, Stephen R. Rannels, Jackie D. Corbin

Bioelectrics Publications

Excluding autophosphorylated species, at least six forms of the regulatory subunit of type II cAMP-dependent protein kinase (R(II)) from various mammalian tissues were identified by sodium dodecyl sulfate (SDS) gel electrophoresis of purified samples and of crude preparations photoaffinity labeled with 8-azido[32P] cAMP and by gel filtration. After autophosphorylation some heart R(II) forms termed type IIA (bovine, porcine, equine, and dog) shifted to a more slowly migrating band on SDS gels while others termed type IIB (rat, guinea pig, rabbit, and monkey) did not detectably shift. Both subclasses of R(II) exhibited variation in apparent M(r) on SDS gels. Bovine and …

Two Classes Of Camp Analogs Which Are Selective For The Two Different Camp-Binding Sites Of Type Ii Protein Kinase Demonstrate Synergism When Added Together To Intact Adipocytes, Stephen J. Beebe, Rob Holloway, Stephen R. Rannels, Jackie D. Corbin

Bioelectrics Publications

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their K(a) for protein kinase and their lipophilicity rather than with either parameters alone. Some of the most potent lipolytic analogs had high I50 values for the particulate low K(m) cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations …

Comparison Of Surface Polysaccharides Of R. Leguminosarum Anu57 (Exo-1), R. Leg. Anu57(Pbr1an) (Nod+, Nif-), And R. Leg. Anu57(Pjb5j1) (Nod+, Nif-), Frank G. Hustmyer

Masters Theses

Extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) are suspected to be involved in the legume/Rhizobium symbiotic process. Polysaccharide compositions from the EPS, LPS, capsular polysaccharide (CPS), and small polysaccharide (SmPS) were compared between Rhizobium leguminosarum ANU57 (parent, Exo^-1) and two mutants R. leg. ANU57(pBR1AN) (nod⁺, nif^-) and R. leg. ANU57(pJB5J1) (nod⁺, nif^-) both containing a Sym (symbiotic) plasmid insertion. R. leg. ANU57(pBR1AN) and R. leg. ANU57(pJB5J1) are nod⁺ on clover and peas, respectively. The nod⁺ mutants produce larger amounts of EPS and decreased amounts of LPS compared to …

Determination Of Erythrocyte Zinc Concentrations Using Zeeman Effect Atomic Absorption Spectroscopy, Tanya Maranda Lewis

Chemistry & Biochemistry Theses & Dissertations

The purpose of this research project was to develop assay procedures for determination of zinc in erythrocytes. Three methodologies were developed. The methods were evaluated on the basis of within-run and between-run precision. The most reproducible method, the 213.856 nm flame methodology, was used to determine red blood cell zinc concentrations in more than 200 samples obtained from the Neonatal Intensive care Unit at the Children's Hospital of the King's Daughters.

This study demonstrated that erythrocyte zinc levels in premature infants were similar to those of healthy adults. A linear relation between plasma and red blood cell zinc concentrations was …

Distribution Of Limonin During The Growth And Development Of Leaves And Branches Of Citrus Paradisi, Cecilia Mcintosh, Richard L. Mansell

Cecilia A. McIntosh