Biochemistry, Biophysics, and Structural Biology Commons^™

A Temperature-Sensitive Mutant Of Escherichia Coli Affected In The Alpha Subunit Of Rna Polymerase, Majid Mehrpouyan

Electronic Theses and Dissertations

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a …

Adsorption To Dipalmitoylphosphatidylcholine Membranes In Gel And Fluid State: Pentachlorophenolate, Dipicrylamine, And Tetraphenylborate, Pavel Smejtek, S. R. Wang

Physics Faculty Publications and Presentations

We measured the dependence of electrophoretic mobility of dipalmitoylphosphatidylcholine (DPPC) vesicles on the aqueous concentration of negatively charged ions of pentachlorophenol (PCP), dipicrylamine (DPA), and tetraphenylborate (TPhB). The objective was to determine how the physical state of hydrocarbon chains of lipids affects adsorption of lipophilic ions. The studies were done at 25 and 42 degrees C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of zeta-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, Ps, and the linear partition coefficient, beta. …

Interaction Between The Component Enzymes Of The Glycine Decarboxylase Multienzyme Complex, David J. Oliver, Michel Neuburger, Jacques Bourguignon, Roland Douce

David J. Oliver

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows …

Mega Doses Of Methotrexate In The Treatment Of Osteosarcoma, M P. Iqbal, I A. Malik

Department of Biological & Biomedical Sciences

Abstract is not provided by the author/publisher

Activation In Vitro Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Panayota Eriotou

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to isolate and purify the enzymes, transfer RNA-guanine ribosyltrasferase and protein kinase C, and to determine whether the phosphorylating enzyme activates the insertion enzyme in vitro. Transfer RNA-guanine ribosyltransferase lost activity within several days after isolation and total, complete reactivation was accomplished in the presence of protein kinase C. This demonstrated that ribosyltransferase's instability is related to the degree of its phosphorylation. It is proposed that modification of tRNA is controlled by protein kinase C. Deactivation of the insertion enzyme leads to hypomodified tRNA which in turn is associated with neoplasia. Potential use …

Laminin Potentiates Differentiation Of Pcc4uva Embryonal Carcinoma Into Neurons, T. M. Sweeney, Roy C. Ogle, C. D. Little

Medical Diagnostics & Translational Sciences Faculty Publications

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by …

Highway Deicing Salts And The Mobilization Of Selected Heavy Metals From Stream Sediments, Harlie David Cole

Dissertations

This study was undertaken to: (a) determine the effect of sodium chloride concentration on mobilizing cadmium, copper, lead, nickel, and zinc from stream sediments; (b) determine if chloro-metal complexes may play a role in any such mobilization; and (c) determine the correlations between chloride ion and each of these metals in an urban stream during winter road-salting periods.

The drainage basin of the Red Run, an urban stream draining all or parts of several cities north of Detroit, Michigan, was chosen as a study site. Stream sediments containing the five heavy metals were stirred with sodium chloride or sodium nitrate …

Proto-Oncogene Regulation By Growth Factors In Bone Cells, Harold Lyndon Merriman

Loma Linda University Electronic Theses, Dissertations & Projects

Previous studies have shown that bone cells in culture produce a number of growth factors that are important in bone regulation. Chick and mouse primary calvarial cultures consist of a mixture of cell types that make it difficult to interpret the results from these model systems. In contrast, MC3T3-E1 cells are a clonal mouse osteoblast-like cell line. Since MC3T3-E1 cells consist of a single population of calvarial cells, they make an ideal system in which to study the autocrine effects of bone growth factors.

Based on this work, MC3T3-E1 cells are now known to produce IGF-I, TGF-beta and IGF-II in …

K-Casein And Its Interaction In Human Milk Micelles, Barbara C. Dev

Loma Linda University Electronic Theses, Dissertations & Projects

Human milk K-casein was isolated from the acid-precipitated casein fraction on Sephadex G-200 and Mono Q HPLC. Samples of K-casein purified from different donors were found similar in both amino acid and carbohydrate composition. A molar extinction coefficient of 11.2 was determined on the basis of amino acid analysis with a norleucine internal standard. Tracer K-casein for human micelle studies was ³H-labeled in the sialic acid moiety of its carbohydrate. The human micelle system was investigated in its native form at 37°C. Micelle reformation by re-equilibration in skimmed milk at 4°C (overnight) followed by 3 h at 37°C was …

Development Of Catalytic Rnas As Potential Anti-Hiv-1 Agents, Pairoj Sae Chang

Loma Linda University Electronic Theses, Dissertations & Projects

RNA molecules with enzymatic activity (ribozymes) targeted to the genome of HIV-1 were developed and demonstrated to mediate site-specific cleavage of the HIV-1 RNA in vitro and to inhibit viral proliferation intracellularly. Two classes of ribozymes that incorporate a "hammerhead" catalytic center were utilized, eotide elements in the viral sequence to provide half of the The first class used nuclcatalytic center. A complementary strand harboring the remaining active site nucleotides was supplied in trans and directed the site-specific cleavage of the HIV-1 RNA target. A second type of ribozyme incorporated the entire catalytic center, minus the site of cleavage, within …

The Use Of Synthetic Oligodeoxyribonucleotides In Direct Analysis Of Single Nucleotide Variations And Inhibition Of Eukaryotic Gene Expression, Daniel Y. Wu

Loma Linda University Electronic Theses, Dissertations & Projects

The present thesis work contributes to two areas of molecular genetics, namely the development of methods for identifying single nucleotides and the understanding of the mechanism by which antisense oligonucleotides inhibit eukaryotic gene expression. Three single nucleotide detection methods are presented. The first method utilizes hybridization with synthetic oligonucleotide probes to detect single or multiple nucleotide differences between alleles in genomic DNA without electrophoretic separation. Total genomic DNA (either digested or undigested with restriction endonucleases) was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. We show that this method can be …

Kinematic Considerations Of Elite Alpine Slalom Ski Racers, David Andrew Goodwin

Masters Theses

This study described specific kinematic parameters associated with success on the international level in alpine slalom ski racing. Sixteen male athletes representing twelve countries comprised the subject pool. Subject selection was based on finish position in the men's special slalom at the 1989 World Alpine Ski Championships in Vail, Colorado. Cinematographical records were analyzed by a method known as direct linear transformation.

The findings of the study indicated that: (a) hip and knee joint angles of the turning leg must approximate 1.80 to 1.83 radians and 1.82 to 2.21 radians, respectively, for efficiency in this turn; (b) a decrease in …

Trace Element Studies On Karachi Populations Part V: Blood Lead Levels In Normal Healthy Adults And Grammar School Children, W W. Manser, R Lalani, S Haider, M A. Khan

Department of Biological & Biomedical Sciences

Blood lead levels of healthy Karachi population were estimated. Mean levels for males, females, soldiers and school children were 34.4, 31.8, 29.9 and 38.2 micrograms/dl respectively. About 93% cases of either sex had elevated lead levels, of whom 30% males and 10% females had levels above the safety limits (40 micrograms/dl). Soldiers living in relatively pollution free area though had levels lower than the rest of the population but 91% had levels over 25 micrograms/dl and only two had acceptable levels. Ninety-two percent children showed levels above 25 micrograms/dl with a large number having levels over 40 micrograms/dl. A very …

Effects Of Chemical Aneuploidogens On Taxol Purified Drosophila And Mouse Brain Microtubules Polymerization And Depolymerization In Vitro, Anil Sehgal

Biological Sciences Theses & Dissertations

The effects of aneuploidogens (aneuploidy causing agents) on taxol-purified microtubules from Drosophila and mouse brain in vitro were studied by using a spectrophotometric assay and electron microscopy. Colchicine, acetonitrile, propionitrile, acrylonitrile, dimethyl sulfoxide (DMSO), griseofulvin and cadmium chloride inhibited microtubule polymerization whereas methoxyethyl acetate (MEA) and methyl mercuric chloride (MMC) did not. All aneuploidogens tested (at 50mM) resulted in reduced rate of elongation of mouse brain microtubules. MMC, cadmium chloride and DMSO resulted in increased rates of Drosophila microtubule elongation whereas the rest of the drugs resulted in decreases. The in vitro results from Drosophila correlate well with the previously …

The Determination Of Clomiphene Citrate And Tamoxifen In Human Blood Plasma And Semen, Janice Irene Kiss

Chemistry & Biochemistry Theses & Dissertations

Triphenylethylene antiestrogen compounds are used to stimulate ovulation in anovulary women and spermatogenesis in oligozoospermatic males. Clomiphene Citrate (CC) is given therapeuticaly as a mixture of the trans (E) and cis (Z) isomers. Each of these isomers has its own characteristic agonist-antagonist profiles. Only the trans form of Tamoxifen (T) is administered therapeutically.

HPLC methodologies were developed for the determination of CC and Tin human blood and seminal plasma. The methodologies involved extraction of the triphenylethylene compounds using hexane, followed by evaporation of the hexane under N₂. The residue was then taken up in a volume of HPLC …

Microwell Based Solid Phase Competitive Enzyme Immunoassay For Estradiol And Estriol, Jeng-Ting Yang

Chemistry & Biochemistry Theses & Dissertations

A sensitive and simple microwell based competition enzyme immunoassay for the quantitative determination of estradiol (E₂) and estriol (E₃) was developed. Nicrowells coated with antibody were incubated with antigen followed by adding horseradish peroxidase (HRPO) conjugate. The assay, which can be performed within two hours at room temperature, involved simultaneous incubation of E₂ - or E₃-HRPO conjugate and serum sample in polystyrene microwells coated with anti- E₂ or anti-E₃ gamma globulin fraction.

Gamma globulin was isolated from whole anti-serum by DEAE-cellulose chromatography. A carbodiimide coupling method was utilized to prepare the …

Development Of A Solid Phase Enzyme Immunoassay For Total Serum Testosterone, Li Li

Chemistry & Biochemistry Theses & Dissertations

An enzyme-labeled immunoassay of sufficient range and sensitivity was developed for serum testosterone determination. In this method, the testosterone in the serum sample and testosterone-HRPO conjugate compete with each other for the limited antibody binding sites on the anti-testosterone gamma globulin coated microwell. The assay can be performed in only 2.5 hours.

The gamma globulin fraction was isolated from the anti-testosterone whole serum by DEAE-cellulose column chromatography. Testosterone-peroxidase conjugate was prepared by a carbodiimide coupling method. The interference which arises from the binding of testosterone to steroid hormone binding protein was eliminated by the addition of 17 beta-estradiol. All assay …

Separation Of Transfer Rna Isoacceptors By Reversed Phase W-Porex C-4 High Pressure Liquid Chromatography, Christine D. Witte

Chemistry & Biochemistry Theses & Dissertations

Transfer RNA (tRNA), is a complex class of small molecules which have been proven to play a major role in protein synthesis. Transfer RNA is composed of 20 families of isoacceptors which may recognize only one particular amino acid. This family of isoacceptors may differ from each other by primary sequence or modified nucleosides. Queuine is a modified nucleoside which is found in the anticodon regions of tRNA isoacceptors for asparagine, aspartic acid, tyrosine and histidine. Queuine has gained the interest of many researchers since queuine deficient tRNA was found in many neoplastic and undifferentiated cell-lines. It is often difficult …

Replication And Amplification Of Defective Interfering Particle Rn As Of Vesicular Stomatitis Virus In Cells Expressing Viral Proteins From Vectors Containing Cloned Cdnas, Asit K. Pattnaik, Gail W. Wertz

School of Veterinary and Biomedical Sciences: Faculty Publications

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have untD now been attained only in ceUs coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle …

Maternal Immunomodulation Of Neonatal Alloantigen Response, Leh Chang

Loma Linda University Electronic Theses, Dissertations & Projects

Remarkable success has been achieved in the transplantation of allogeneic cardiac grafts into newborn infants at Loma Linda University Medical Center. The superior graft survival rate documented in these patients has not correlated with the degree of immunosuppression rendered, or the selection of genetically matched donors. However, the clinical success has correlated with the age of the recipient at the time of receiving a transplant. Patients receiving an allograft within the first few weeks of life are unique in that they seem to accept the alloantigens of their cardiac graft while responding aggressively to antigens in their environment. These observations …

Purification And Characterization Of Enoyl-Acp Reductase From Euglena Gracilis, Margie M. Tucker

Electronic Theses and Dissertations

Enoyl-(acyl-carrier-protein) reductase was purified from the phytoflagellate Euglena gracilis. Its purification employed DEAE-Sephacel chromatography, Matrex Orange chromatography, and affinity chromatography using acyl carrier protein (ACP) covalently bound to Sepharose as the affinity ligand. Matrex Orange chromatography resolved two different enoyl-ACP reductases having different characteristics. Euglena gracilis appears to resemble higher plants in the possession of two isoforms of this enzyme. Antibodies specific for the cofactor binding site of NADP (H)-requiring dehydrogenases were obtained. They were isolated from a polyclonal population of antibodies directed against yeast glucose-6-phosphate dehydrogenase by affinity chromatography using chicken liver malic enzyme as the affinity ligand. The …

Energetic Requirements For Bacterial Protein Export, John Andrew Corbett

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bacterial protein export involves the translocation of precursor proteins across the inner cytoplasmic membrane. Over 100 proteins are exported from Escherichia coli. This study showed that energy in the form of ATP and membrane gradient energy is essential for the export of leucine specific binding protein and β-lactamase precursors. Ionophores or combinations of ionophores (SF6847, valinomycin/nigericin and valinomycin/monensin) which dissipate protonmotive force inhibit protein export. Valinomycin alone also inhibits export, but not as well as reagents which dissipate protonmotive force. Nigericin or monensin alone slightly stimulate protein export. These results suggest that the transmembrane electrical potential (∆Ψ) is the …

The Role Of The Voltage Gradient In The Agarose Gel Electrophoresis Of Dna, David Wheeler

Theses and Dissertations in Biomedical Sciences

In Part I of this dissertation, empirical equations for predicting DNA mobility during agarose gel electrophoresis (AGE) from voltage gradient are developed from the data of McDonnel (36) for electrophoresis in a 1.6% agarose gel. These equations represented the data well for DNA between 2 and 10 kilobase pairs (KBp) in length. A computer program, called GELSIM, which incorporates these equations is described in Part II. GELSIM was designed to allow researchers to analyze electrophoresis data by predicting the effect on DNA migration of altering the voltage of electrophoresis. In this way, electrophoretic banding patterns produced using different voltages could …

Thermodynamic Extent Of Counterion Release Upon Binding Oligolysines To Single-Stranded Nucleic Acids., David Mascotti, Timothy Lohman

David P. Mascotti

A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants--i.e., (delta log Kobs/delta log[K+])--over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous …

Nucleotide Sequence And Further Characterization Of The Synechococcus Sp. Strain Pcc 7002 Reca Gene: Complementation Of A Cyanobacterial Reca Mutation By The Escherichia Coli Reca Gene, R C. Murphy, G E. Gasparich, D A. Bryant, R D. Porter

Gail Gasparich

Cathepsin B: Nrna Characterization, Gene Structure And Expression In Rodent Normal Tissues And Malignant Tumors With Different Metastatic Potentials, Fang Qian

Dissertations

No abstract provided.

Enzymatic And Nonenzymatic Mechanisms For Ferric Leghemoglobin Reduction In Legume Root Nodules, Manuel Becana, Robert V. Klucas

Department of Biochemistry: Faculty Publications

Evidence is presented for the operation in nodules of at least four systems for restoring functional ferrous leghemoglobin (Lb²⁺) from its inactive, ferric form. (i) Reduction of ferric leghemoglobin (Lb³⁺) by a reductase. The enzyme is a flavoprotein of 100 kDa with two equally sized subunits and exhibits a K_m of 9 µM for soybean Lb³⁺ component a and a K. of 51 µM for NADH. NADPH is only 30% (initial velocities) as effective as NADH. Lb³⁺ reductase converts 215 nmol of Lb³+to Lb²⁺ •CO (or Lb²⁺ •O_{2 …}

A Retinoic Acid Responsive Gene, Mk, Produces A Secreted Protein A With Heparin Binding Activity, Masaru Nakamoto

Biology Faculty Publications

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the S-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.

Isoprenylation Is Required For The Processing Of The Lamin A Precursor, Michael Sinensky, L. A. Beck, T. J. Hosick

Michael Sinensky

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. …

Isoprenylation Is Required For The Processing Of The Lamin A Precursor, Michael Sinensky, L. A. Beck, T. J. Hosick

Faculty Publications, Biological Sciences

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. …