Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

Some Diazinon Analogues Containing The 4-Trifluoromethyl Group / Herman Gershon, Donald D. Clarke, Anthony T. Grefig, And Thomas E. Anderson Boyce Thompson Institute For Plant Research At Cornell University, Ithaca, Ny 14853, U.S.A. Department Of Chemistry, Fordham University, Bronx, Ny 10458, U.S.A., Herman Gershon, Donald Dudley Clarke Phd, Anthony T. Grefig, Thomas E. Anderson

Chemistry Faculty Publications

Diazinon analogues were prepared containing trifluoromethyl in place of the 4-methyl group and methylthio (2a), amino (2b), dimethylamino (2c), methylphenylamino (2d), or isopropyl (2e) in position 2 of the pyrimidine ring. The most active analogue (2 b) was less than half as insecticidal as Diazinon

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. …

Conformational And Circular Dichroism Studies On N-Acetyl-L-Prolyl-D-Alanyl-Methylamide, S. Ramaprasad

Journal of the Arkansas Academy of Science

The compact ordered conformations of the molecule N-Acteyl-L-Prolyl-D-Alanyl-Methylamide have been studied by semiempirical energy calculations in vacuum and circular dichroism (CD) in solution. The presence of ordered structure has been observed in hydrogen bond promoting solvents like trifluoroethanol by CD studies. In hydrogen bond breaking solvents, like trifluoroacetic acid (TFA), significant fraction of the ordered conformers probably assume extended conformation without intramolecular hydrogen bonds and perhaps are in equilibrium with the fraction of compact ordered structures. The trend observed in going from nonpolar to polar solvent is also compatible with the previous NMR studies in solution. The semiempirical energy calculations …

Estuarine Studies : An Activities Text For Maine Schools, Education Division - Department Of Marine Resources

Maine Collection

Estuarine Studies : An Activities Text for Maine Schools

Fisheries Education Unit #16 - Produced by The Education Division, Department of Marine Resources, State House Station 21, Augusta, Maine 04333-0021.

Revised 1990. Printed Under Appropriation No. 3140.3260.

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.

Primary Sequence Of The Escherichia Coli Fadba Operon, Encoding The Fatty Acid-Oxidizing Multienzyme Complex, Indicates A High Degree Of Homology To Eucaryotic Enzymes, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in α₂β₂ conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming …

The Role Of Small Peptides In Cancer Physiology And Chemotherapy, Bao-Ling Tsay

Theses and Dissertations in Biomedical Sciences

The targeting of proven anticancer drugs specifically to cancer cells would provide a unique opportunity to restrict neoplasms without damaging the cancer patient. The present research utilizes the phenomenon of illicit transport, i.e. the coupling of normally impermeant metabolites to permeant metabolites, in targeting the drug melphalan to mouse Ehrlich ascites tumor cells. The dipeptide beta-alanyl-melphalan was synthesized and tested in vitro for toxicity towards mouse Ehrlich ascites tumor cells, mouse liver cells, and mouse 3T3 embryonic cells. The parent compound, melphalan, was used as a control treatment. In addition, both melphalan and beta-alanyl-melphalan were utilized in in vivo chemotherapeutic …

Mermithid Nematodes: Sem Observations Comparing Hexamethyldisilazane And Critical Point Drying Methods, William R. Bowen, Christopher C. Hemann, Arthur A. Johnson, Barry H. Good

Journal of the Arkansas Academy of Science

Morphological features of mermithid nematodes (Mermithidae) were studied with scanning electron microscopy, using hexamethyldisilazane-air drying in comparison with critical point drying via liquid carbon dioxide. Although general morphologic preservation of both HMDS-dried and CP-dried specimens was similar, structural features of the complex cuticle and internal organization were more easily resolved at higher magnifications in the HMDS-dried nematodes. These features include the superficial cuticular annulations, the fibrillar inner cuticle and peg-like microtrabeculae. The previously undescribed microtrabeculae are of special interest since they may facilitate an interaction of the mermithid (and perhaps nameatodes in general) musculature with its body wall that, at …

The Determination Of Tamoxifen Citrate In Human Blood Serum And Semen, Karimdad Tahmoresi

Chemistry & Biochemistry Theses & Dissertations

High performance liquid chromatography (HPLC)methodologies for the determination of tamoxifen and its metabolites 4-hydroxytamoxifen and n-desmethyltamoxifen, and using cis-tamoxifen as an internal standard have been developed.

Clinical analyses of tamoxifen and its metabolites in human serum and semen, were performed as follows: The analytes were extracted from serum and semen with hexane, the hexane evaporated, the dried extract then reconstituted with methanol, followed by reverse phase high performance liquid chromatography.

Between-run coefficients of variation (CV'S) for tamoxifen and n-desmethyltamoxifen for serum sample analyses, (n = 15) were, 4% and 13% at mean (SD) concentration of 140.2 (6.2) and 71.8 (9.8) …

Regulation Of Queuine Insertion Into Transfer Rna: Effects Of Tumor Promoters, Bonnie Jean Brooks

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to treat normal human fibroblasts with known tumor promoters and observe queuine modification of tRNA. The queuine insertion enzyme, tRNAguanine ribosyltransferase, was studied in vivo and in vitro. Tumor promoter-treated human fibroblast cultures exhibited variable queuine-insertion rates with a transient inhibition that correlated with variable levels of queuine modified tRNA over time in culture from passages 3 — 8. In contrast the in vitro studies showed that phorbol cetera and saccharin actually increased insertion with strong evidence indicating phosphorylation as a positive modulating force of the enzyme activity. It is proposed that chronic stimulation …

Dietary Protein Deficiency And Mycobacterium Bovis Bcg Affect Interleukin-2 Activity In Experimental Pulmonary Tuberculosis, David N. Mcmurray, C. L. Mintzer, R. A. Bartow, Rebecca D. Parr

Faculty Publications

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and …

A Comparison Of Chemical Composition & Fermentation Patterns Of Alternative Silages To Whole Plant Corn Silage, Susan Fox

Masters Theses & Specialist Projects

A comparison of the relationship of fermentation to chemical composition was made for forages which were wilted and ensiled at 35 to 45 percent dry matter. Trial I consisted of three forages ensiled in October, 1981: interseeded soybeans and grain sorghum. whole plant corn with added anhydrous ammonia, and whole plant corn with shelled corn added at a rate of 150 kg/t of fresh forage. Trial II consisted of two forages ensiled in 1982: interseeded soybeans and pearlmillet, and wheat. Temperatures of fermentation were collected, and chemical composition during the first 25 days of fermentation analyzed.

Production data were also …

Spectroscopic Study Of Decomposition Products Of Gossypol, David Bernard Hankins

Chemistry & Biochemistry Theses & Dissertations

Basic research into reproductive physiology has begun to identify possible new approaches to controlling fertility. Gossypol, a natural polyphenol compound isolated from cottonseed oil, has been shown to be an effective male antifertility agent. The decomposition products of gossypol which are thought to possess more potent antifertility effects than the parent compound gossypol have been studied extensively in more recent research. The purpose of this research is to develop techniques and methods which could be used to identify decomposition products of gossypol. A model decomposition reaction by which a smaller molecule can be isolated in a pure state has been …

Simultaneous Determination Of 3-Methoxy-4-Hydroxyphenyl-Glycol, Homovanillic Acid, And 5-Hydroxyindoleacetic Acid In Plasma By High Performance Liquid Chromatography, Yung-Te Tseng

Chemistry & Biochemistry Theses & Dissertations

A rapid, reliable, and sensitive method for simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), Homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5- HIAA) in plasma is described. The method is based on high performance liquid chromatography with electrochemical detection. Samples were filtered with Amicon Centrifree micropartition systems and extracted with organic solvent before chromatography. Chromatographic separation of sample components was achieved isocratically in a C18 reverse-phase column. Quantitation was performed by measuring the peak height ratios of the sample components to an internal standard. Analytical recoveries ranged from 50.78 (SD 4.2%) to 67.94 (SD 3.28). Within-run coefficient of variations (CVs) ranged from 3.78 …

Microwell Based Competitive Enzyme Immunoassay For Quantifying Serum Triiodothyronine And Thyroxine Levels, Yiwen Yang

Chemistry & Biochemistry Theses & Dissertations

A microwell based competitive enzyme immunoassay for determination of total thyroxine and triiodothyronine in human serum was developed with use of horseradish peroxidase conjugate. The assay, which can be performed in only 1.5 hours at room temperature, involves simultaneous incubation of T₃- or T₄- HRPO conjugate and serum sample in anti-T₃ or anti-T₄ gamma globulin fraction coated polystyrene microwells.

Isolation of anti-T₃ and anti-T₄ gamma globulin fractions were accomplished from whole antiserum by using DEAE-cellulose chromatograph. A carbodiimide coupling method was described to prepare the T₄- and T₃-HRPO …

Regulation Of Epidermal Growth Factor Receptor Synthesis By Retinoic Acid And Dexamethasone, Kerby C. Oberg

Loma Linda University Electronic Theses, Dissertations & Projects

Fetal rat lung (FRL) cells incubated in the presence of retinoic acid or dexamethasone exhibit altered ¹²⁵I-epidermal growth factor (EOF) binding capacity. Retinoic acid enhances ¹²⁵I-EGF binding 3-fold following 24 hours of 37° incubation. In contrast, FRL cells incubated in the presence of dexamethasone display a 2-fold reduction in ¹²⁵I-EGF binding capacity. The change in binding capacity, induced by either hormone, was due to alterations in the average number of receptors per cell as determined by Scatchard analysis of ¹²⁵I-EGF binding. In the absence of retinoic acid or dexamethasone, FRL cells were calculated to have approximately …