Molecular Biology Commons^™

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Characterization Of Human T-Cell Leukemia Virus 1 Reverse Transcriptase, Pinky Gundayao Agbuya

Theses and Dissertations in Biomedical Sciences

Human T-cell leukemia virus 1 (HTLV-1) is a type C human retrovirus which has been the causative agent of Adult T-cell leukemia. Replication of the retrovirus; requires a reverse transcriptase which converts the retroviral RNA into DNA which is later incorporated into the host's genome. Very little is known about the reverse transcriptase of HTLV-1. Researchers have attempted to purify HTLV-1 RT by isolating the enzyme from human cell lines. Because large amounts of protein could not be produced by this isolation method, the reverse transcriptase cannot be fully characterized. In this research, a recombinant protein expressed in E. coli …

Absence Of Insect Juvenile Hormones In The American Dog Tick, Dermacentor Veriabilis (Say) (Acari: Ixodidae), And In Ornithodoros Parkeri Cooley (Acari: Argasidae), P. A. Neese, Daniel E. Sonenshine, V. L. Kallapur, C. S. Apperson, R. M. Roe

Biological Sciences Faculty Publications

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with _L-[methyl-³H]methionine and farnesoic acid or with [1-¹⁴C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12–168 …

Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression And Hormone Regulation In Human Placental Jeg-3 Cells, Heming Zheng

Theses and Dissertations in Biomedical Sciences

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, I studied the mechanisms of the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The results showed that all of the previously identified four elements are required for the full activity of the hGnRH upstream promoter in JEG-3 cells, while the element 4 (FP4, −987/−968) is the most important. Studies performed with 5′ end deletion of this region confirmed these observations. Further, supershift assay using Oct-1 antibody demonstrated the involvement of Oct-1 in the FP4 DNA-protein interaction in JEG-3 …

Tissue-Specific Expression And Steroid Hormone Regulation Of Human Gonadotropin -Releasing Hormone (Hgnrh) Gene In Placental Cells (Jeg -3 Cells), Zhengguang Chen

Theses and Dissertations in Biomedical Sciences

Using JEG-3 cells as an in vitro model, I investigated the mechanisms behind the tissue-specific expression and steroid hormone regulation of the hGnRH gene in the human placenta. The hGnRH upstream promoter was found to be functionally active in JEG-3 cells. The DNA sequence responsible for functioning of the upstream promoter in JEG-3 cells is narrowed to a region between –1048 bp and –730 bp. This DNA fragment contains four elements, which can bind with nuclear extract from JEG-3 cells (but not from GT1-7 cells).

Estradiol (E2) represses the hGnRH upstream promoter activity in JEG-3 cells. This inhibition is receptor-mediated, …

Production Of Copper-Complexing Ligands In Response To Elevated Concentrations Of Copper In Marine Synechococcus Spp., Arunsri Chatkaeomorakot Brown

Theses and Dissertations in Biomedical Sciences

Synechococcus spp. (marine cyanobacteria) are extremely sensitive to copper toxicity and can produce high-affinity ligands of unknown structure which form complexes with free cupric ion. These ligands may contribute to the biological control of the levels of free cupric ions in surface seawater. Synechococcus spp. are known to produce metallothioneins (MT) in response to cadmium and zinc stress. In the present study the hypothesis that marine Synechococcus produce MT in response to copper was tested. Three marine Synechococcus spp. i.e., PCC 7001, PCC 73109, and PCC 7003, were exposed to different concentrations of CUSO 4 for various time periods. Size …

Transcriptional Regulation Of The Bmp2 Gene: Retinoic Acid Induction In F9 Embryonal Carcinoma Cells And Saccharomyces Cerevisiae, Loree C. Heller, Yong Li, Kevin L. Abrams, Melissa B. Rogers

Bioelectrics Publications

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays …

Construction Of An Escherichia Coli Lac Repressor-Based System To Study Human Cytomegalovirus Gene Expression, Laura Fernanda Cageao-Luchetti

Theses and Dissertations in Biomedical Sciences

An Escherichia coli lac repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the lac repressor was generated (RVlac), and an HCMV-specific promoter was targeted for conditional expression by inserting the lac operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter …

Expression, Isolation And Purification Of Human Zona Pellucida Protein 3, Ting-Fung Chi

Theses and Dissertations in Biomedical Sciences

Mammalian fertilization involves interactions of sperm surface receptors with the ligands of the zona pellucida, an extracellular matrix surrounding the ovulated oocytes. In humans, the zona pellucida is composed of three major glycoproteins. One of them, ZP3, participates in the primary sperm binding and in the subsequent triggering of the spermatozoa's acrosome reaction. Studies on the role of this specific protein in the human fertilization process are hampered by the limited amount of available biologically functional proteins.

By use of a pcDNA 3.1(+) expression vector, a transfecting-vector was constructed containing a 1.3 kb histidine tagged hZP3 cDNA. This histidine tagged …

Characterization Of The Biological Functions Of Human Recombinant Zona Pellucida Protein 3, Yu Wen Juan

Theses and Dissertations in Biomedical Sciences

Recombinant human zona pellucida protein 3 (rhZP3), expressed. isolated and purified from PA-1 cells, is characterized for its biological activity and the role in the signal transduction pathway. Characterization of the biological activity of rhZP3 was detected by hemizona assay and immunofluorescence staining of acrosome reaction The results indicated that rhZP3 exhibited an inhibition in the binding assay (HZI 43.6 +/-3.3; n = 9; 30 ng/mL rhZP3) and induction of acrosome reaction (198.6% +/- 77.2% increase from baseline; n = 29; 30 ng/mL rhZP3). It was further confirmed by the transmission electron microscopy that there was no difference in morphology …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

Type I Camp-Dependent Protein Kinase Delays Apoptosis In Human Neutrophils At A Site Upstream Of Caspase-3, Lav K. Parvathenani, E. Stephen Buescher, Enrique Chacon-Cruz, Stephen J. Beebe

Bioelectrics Publications

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had …

Evaluation Of Toxicity, Bioavailability And Speciation Of Lead, Zinc And Cadmium In Mine/Mill Wastewaters, Mujde Erten-Unal, Bobby G. Wixson, Nord Gale, Jerry L. Pitt

Civil & Environmental Engineering Faculty Publications

The toxicity of common compounds of lead, cadmium and zinc was evaluated in waters similar to that found in the world's largest lead producing area in Missouri. Static, acute toxicity tests were performed using fathead minnows (Pimephales promelas) and water fleas (Daphnia magna) respectively. Test organisms were subjected to varying amounts of sulfide, carbonate, chloride and sulfate salts of lead, zinc and cadmium mixed in hard, alkaline waters typical to this region. Median lethal concentrations were calculated using nominal versus measured metal concentrations. Measured metal concentrations included four different metal fractionation (extraction/filtration) techniques at different pH …

Sex Chromosome Ratios And Aneuploidy Rates In The Zona Pellucida Selected Spermatozoa From Normal And Subfertile Men, Qinuo Fan Van Dyk

Theses and Dissertations in Biomedical Sciences

The human hemizona has been demonstrated to select spermatozoa with good motility, normal morphology, and the capacity to undergo the zona-induced acrosomal reaction. The studies conducted here are directed at using the human hemizona to investigate two key questions: (1) whether human X-chromosome bearing spermatozoa (X-sperm) and Y-chromosome bearing spermatozoa (Y-sperm) differ in their functional survival time (assessed by their capacity to bind to the human zona pellucida) after prolonged in vitro incubation, and (2) whether hemizona-bound spermatozoa have a reduced aneuploidy rate compared to unbound (and therefore, unselected) spermatozoa?

In the functional survival study, donor spermatozoa were held in …

Type Ii Estrogen Binding-Site Agonist: The Synthesis And Biological Evaluation Of The Enantiomers Of Methyl-Para-Hydroxyphenyllactate (Mehpla), Lester G. Pretlow

Theses and Dissertations in Biomedical Sciences

The function of the type II estrogen binding site (EBS) has yet to be determined. However, a high affinity ligand for the binding site has been identified through HPLC and GC-MS. This ligand, MeHPLA, bears a structural relationship to a group of compounds called "phytoestrogens" which, along with MeHPLA, have been observed to suppress the cellular proliferation of estrogen sensitive MCF-7 breast cancer cells in vitro. Additionally, MeHPLA has been observed to suppress the growth of rat uteri in vivo. The high affinity of MeHPLA for the type II EBS suggests that this interaction is responsible for the observed suppression …

Interaction Of Acth And Estradiol In The Regulation Of Growth, Differentiation, And Steroidogenic Maturation Of The Baboon Fetal Adrenal Gland, Maria Gomez Leavitt

Theses and Dissertations in Biomedical Sciences

The present study determined whether the growth of, as well as ACTH receptor and P-450 enzyme messenger ribonucleic acid and/or protein levels in the baboon fetal adrenal are dependent upon fetal pituitary ACTH during mid and late gestation and the mechanism by which placental estrogen modulates ACTH actions. Administration of betamethasone (3mg/day) to baboon mothers on days 60-99 of gestation and to the fetus (0.6 mg/, n = 4) or to the fetus (0.6mg) and mother (6 mg/ml; n = 4) every other day between days 150-164 of gestation (term = 184 days) decreased (P $53β-hydroxysteroid dehydrogenase-isomerase (3β-HSD(+)ctochrome P450 17α-hydroxylase, …

Fluorescence In Situ Hybridization Analysis Of Human Embryos Derived From In Vitro And In Vivo Matured Oocytes, Constance Descisciolo

Theses and Dissertations in Biomedical Sciences

Despite adequate hormonal stimulation, oocytes collected for the purpose of in vitro fertilization and embryo transfer display several levels of nuclear maturity. Preovulatory or mature oocytes, technically those that are Metaphase I or II, are inseminated shortly after aspiration and assessed for fertilization the following day. Prophase I oocytes, also called germinal vesicle-bearing or immature oocytes, require a 24-36 hour period in culture before being exposed to spermatozoa. During this time, the majority of Prophase I oocytes complete nuclear maturation in vitro, progressing from germinal vesicle breakdown through first polar body extrusion. If inseminated, many in vitro matured oocytes fertilize …

The Cellular And Molecular Dynamics Of The Queuosine Modification In Transfer Rna: Definition, Modulation, Deficiencies And Effect Of The Queuosine Modification System, Rana C. Morris

Theses and Dissertations in Biomedical Sciences

The presence of the queuosine modification in the wobble position of tRNA^asn, tRN^asp, tRNA^his, and tRNA^tyr is associated with a decrease in cellular growth rate, an increase in the ability to withstand environmental stress, and differentiation of pleuripotent cells into mature phenotypes. The loss of this normal modification is strongly correlated with neoplastic transformation and tumor progression of a wide variety of cancers.

The "normal" system for formation of the queuosine modification in tRNA was studied in human fibroblast cell cultures and in mouse, rat and human liver tissues. The queuosine modification system …

Cγ- And Cα-Subunits Provide The Specificity For Camp/Pka Function, Wei Qing Zhang

Theses and Dissertations in Biomedical Sciences

The Cα and Cγ of the cAMP-dependent protein kinase (PKA) are two highly homologous (83% amino acid identity), yet functionally distinct isozymes in vitro for substrate and pseudosubstrate specificity, and in intact cells for cell phenotypes (Beebe, 1992).

To determine the molecular mechanisms underlying the distinct functions in cell phenotypes, in vitro experiments were designed to make a detailed comparison of Cγ and Cα for substrate and pseudosubstrate specificity. To this end, Cγ and Cα were expressed m mammalian cells, bacteria and Sf9 insect cells using baculovirus. Abundant expression of active enzyme was cell system specific. Cγ and Cα expressed …

Abnormalities In Post-Translational Processing Of Platelet Rap 1b In Niddm: A Possible Cause Of Platelet Hyperactivity And Cardiovascular Disease In Diabetes, Elizabeth Ann Hall

Theses and Dissertations in Biomedical Sciences

Post-translational processing is critical for the appropriate subcellular localization and function of platelet G-proteins. The majority of the platelet responses to agonists are mediated through specific receptor/G-protein complexes. Therefore, G-protein activity is central to "normal" platelet activity (i.e. aggregation). We have shown that Simvastatin, the in vivo inhibitor of HMG CoA Reductase and therefore isoprenoid synthesis, inhibits the post-translational processing of specific platelet G-proteins and alters platelet responses to agonists. These results show the importance of post-translational processing of G-proteins to platelet activity. Altered post-translational processing of specific G-proteins may explain platelet hyperactivity and the increased incidence of cardiovascular disease …

The Drosophila Melanogaster Rad54 Homolog, Dmrad54, Is Involved In The Repair Of Radiation Damage And Recombination, Rolf Kooistra, José B. M. Zonneveld, Anja De Jong, Jan C. J. Eeken, Chris J. Osgood, Jean-Marie Buerstedde, Paul H. M. Lohman, Albert Pastink

Biological Sciences Faculty Publications

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females …

Modulation Of Queuine Uptake And Incorporation Into Trna By Protein Kinase C And Protein Phosphatase, Rana C. Morris, Bonnie J. Brooks, K. Lenore Hart, Mark S. Elliot

Chemistry & Biochemistry Faculty Publications

It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that …

Biochemical And Molecular Characterization Of The Prostate-Specific Membrane Antigen (Psma), John Karl Troyer

Theses and Dissertations in Biomedical Sciences

Prostate cancer is the most common malignancy and the second leading cause of cancer death in males in the United States. Additionally, the number of deaths attributed to prostate cancer is increasing at a rate of approximately 8% a year. Development of new diagnostic and therapy strategies are needed in order to improve the life expectancy of patients with this disease. One tool which may allow for improvements in prostate cancer diagnosis and therapy is the monoclonal antibody (MAb) 7E11-C5.3 which was first described in 1987. Since then, the antigen recognized by MAb 7E11-C5.3 has been named the prostate specific …

Investigation Of The Substrate Recognition Characteristics And Kinetics Of Mammalian Mitochondrial Dna Topoisomerase I, Zeki Topcu

Theses and Dissertations in Biomedical Sciences

Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. …

Identification And Characterization Of Mitochondrial Dna Variants In Alzheimer's Disease, Natasha Singh Hamblet

Theses and Dissertations in Biomedical Sciences

Alzheimer's Disease (AD) is a complex neurodegenerative disorder that affects a significant portion of the human population regardless of ethnicity or gender. A mitochondrial hypothesis of AD has been proposed based on a number of studies which establish altered oxidative phosphorylation (OXPHOS) and ATP synthesis in AD tissue. ATP demand is most prevalent in the brain; damage to OXPHOS could severely impair brain metabolism, thereby leading to a decline in cognitive function. Four out of five complexes in the OXPHOS pathway are partly encoded by mitochondrial DNA (mtDNA); thus, this may be a crucial site of lesions that alter brain …

In Vitro Expression Of Human Zona Pellucida Protein 3, Chih-Wei Chen

Theses and Dissertations in Biomedical Sciences

Human zona pellucida protein 3 (hZP3) is the putative receptor on the zona pellucida of the mature oocyte that recognizes and binds sperm, and therefore plays a critical role in fertilization.

A full length cDNA of hZP3 (1278 bp) was amplified from the human ovary mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The hZP3 cDNA was subcloned into PSK and pREP4 expression vectors. The cDNA of hZP3 was further characterized by restriction mapping, PCR, auto-sequencing and Southern blot analysis by using an internal oligonucleotide probe, and found to be identical to the one reported by J. Dean. Using autosequencing, 289 …

Identification And Characterization Of Genes Associated With V-Jun Induced Cell Transformation, Martin Toralballa Hadman

Biological Sciences Theses & Dissertations

The v-jun oncogene was initially identified as the causative agent for fibrosarcomas in chickens. Studies show that overexpression of v-Jun proteins transforms chicken embryo fibroblasts (CEF) in vitro, and forms tumors in chickens in vivo. The mechanisms for this are not clearly defined. Conceivably, overexpression of an unregulated transcription factor would cause cell transfonnation by illicit regulation of its target genes. In support of this, we show that in vivo v-Jun complexes exhibit differential binding to in vitro generated AP-1 and 'AP-1 like' target sequences, suggesting that the pattern of target gene expression is altered during cell transformation. …

The Kinetic Characterization Of Mutant L289p Of Recombinant Hiv-1 Reverse Transcriptase, Tryn Thomas Stimart

Chemistry & Biochemistry Theses & Dissertations

The Human Immunodeficiency Virus, type 1 (HIV-1) is the causative agent of the Acquired immunodeficiency Syndrome (AIDS). Currently, AIDS is the leading cause of death amongst young people in the United States. The large increase of HIV-1 infections over the past decade has spawned considerable research in fighting the spread of this disease. One area of intense HIV-1 research has focused on the enzyme Reverse Transcriptase (RT) which is an essential enzyme involved in the replication and life cycle of HIV-1. HIV-1 RT is composed of p66 and p51 subunits and is only active as a heterodimer in vivo. …

Pathogenicity Of Murine Cytomegalovirus Mutants, Victoria Jean Cavanaugh

Theses and Dissertations in Biomedical Sciences

The purpose of this study was to identified nonessential murine cytomegalovirus (MCMV) genes involved in pathogenesis in vivo. Our approach to identifyjng these genes consisted of constructing MCMV mutants, and then analyzing these mutants in vitro and in vivo. Recombinant viruses (RV) expressing the β-glucuronidase marker gene were constructed by site-directed insertion and deletion mutagenesis of the MCMV Hind III-J and -I regions of the viral genome. Mutations were targeted to this region of the MCMV genome because the corresponding region of the human CMV genome is nonessential and is involved in down-regulating major histocompatibility complex (MHC) class I expression …