Cell and Developmental Biology Commons^™

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 …

Telomerase Activity In Human Preimplantation Embryos, Diane Lynne Wright

Theses and Dissertations in Biomedical Sciences

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells, which stabilizes the telomeres and avoids cellular senescence. Telomerase has been identified in various embryonic cell stages, hematopoietic cells, and in >85% of tumor tissue biopsies analyzed. The ability to measure the potential to proliferate successfully could provide an objective measure of an embryo's quality. The objectives of this study were to modify the telomeric repeat amplification protocol (TRAP) assay system for increased sensitivity to allow detection of telomerase activity in the single cell of an oocyte and embryo, obtain telomerase activity levels for the oocyte …

Use Of Extractable Lipofuscin For Age Determination Of Blue Crab Callinectes Sapidus, Se-Jong Ju, David H. Secor, H. Rodger Harvey

OES Faculty Publications

The blue crab Callinectes sapidus is an economically and ecologically important species in many temperate estuaries, yet stock assessments have been limited to length-based methods for demographic analyses. We evaluated the potential of age pigments (lipofuscins) sequestered in neural tissue of eye-stalks and brains to estimate the age of blue crabs collected from Chesapeake Bay and Chincoteague Bay. The rate of lipofuscin accumulation was determined using crabs of known age reared in the laboratory. Age pigments were extracted from neural tissues (eye-stalk or brain), quantified, and normalized to protein content to allow comparisons across tissue types and crab sizes. Field-collected …

Identification And Characterization Of Determinants Of Head And Neck Tumor Cell Invasion, Yangguan Wu

Theses and Dissertations in Biomedical Sciences

Head and neck squamous cell carcinoma (HNSCC) is a common malignant disease with poor prognosis. The majority of patients die from local invasion or lymphatic metastasis. The mechanism(s) underlining the invasiveness of HNSCC are poorly understood. Utilizing a panel of HNSCC cell lines previously established in our laboratory, we tested the application and relevance of the three-step hypothesis of tumor invasion to HNSCC and investigated the mechanism(s) pertaining to the regulation of each step in the invasive process. Data presented in this thesis demonstrated that tumor cell invasion in HNSCC is a complex process involving three repeated sequential steps: adhesion, …

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, …

Insulinlike Growth Factor 1- And 2-Augmented Collagen Gel Repair Of Facial Osseous Defects, James S. Toung, Roy C. Ogle, Raymond F. Morgan, William H. Lindsey

School of Medical Diagnostics & Translational Sciences Publications

BACKGROUND: Defects of the facial bone structure are common problems for the facial plastic surgeon. Native type 1 collagen gels (T1CGs) have been shown to mediate repair of facial critical-size defects in rat models.

OBJECTIVE: To evaluate the efficacy of T1CG augmented with insulinlike growth factor (IGF) 1, IGF-2, and a combination of IGF-1 and IGF-2 on the repair of facial critical-size defects in a rodent model.

METHODS: Twenty-four retired male breeder Sprague-Dawley rats were divided into 4 groups of 6 animals. Facial critical-size defects were created by removing the nasalis bones with a bone-cutting drill. Defects were treated with …

Efficacy Of AllowashTm Solution In Solubilization Of Bone Marrow, Alyce Marie Linthurst

Biological Sciences Theses & Dissertations

LifeNet currently cleans allograft bone utilizing Allowash^TM Technology, which encompasses the use of a closed continuous circulation chamber to remove bone marrow by flushing it with Allowash^TM Solution for a prescribed time and temperature after which, it is rinsed with various solutions to remove residual detergent that could be toxic to the recipient. A portion of this process employs Allowash^TM Solution to improve the solubilization and removal of protein and cellular elements in the red bone marrow. This study examines the effectiveness of Allowash^TM Solution in comparison to its component detergents Nonidet P40, Nonoxynol 9 and …

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 …

Reaction-Diffusion Models Of Cancer Dispersion, Kim Yvette Ward

Mathematics & Statistics Theses & Dissertations

The phenomenological modeling of the spatial distribution and temporal evolution of one-dimensional models of cancer dispersion are studied. The models discussed pertain primarily to the transition of a tumor from an initial neoplasm to the dormant avascular state, i.e. just prior to the vascular state, whenever that may occur. Initiating the study is the mathematical analysis of a reaction-diffusion model describing the interaction between cancer cells, normal cells and growth inhibitor. The model leads to several predictions, some of which are supported by experimental data and clinical observations $\lbrack25\rbrack$. We will examine the effects of additional terms on these characteristics. …

Pressure-Induced Intracellular Signaling In Isolated Arteries, Victor A. Miriel

Theses and Dissertations in Biomedical Sciences

The ability of cells to respond to mechanical stimuli has been studied through a variety of techniques in numerous cell types. The cells of the vascular wall have adapted to specific mechanical stresses through the activation of intracellular signaling pathways which result in cell-specific responses such as hypertrophy, hyperplasia, proliferation, and migration. Vascular smooth muscle of the arteries have been shown to be sensitive to mechanical stimuli such as stretch, and pressure.

This study attempts to add to the current knowledge of mechanotransduction by utilizing the isolated artery preparation. This preparation allows for the study of vascular smooth muscle signal …

The Role Of Glycoconjugates In Mediating Human Fertilization And Induction Of Fetomaternal Tolerance, Manish S. Patankar

Theses and Dissertations in Biomedical Sciences

Using the hemizona assay (HZA), a in vitro sperm-egg binding assay, we show that specific glycoconjugates known to inhibit immune cell interactions mediated by the selectins, potently block human sperm-egg binding. The selectin ligand sialyl Lewis^x inhibits sperm binding in the HZA by 60% at a concentration of 1 mg/ml. Our data indicates that glycodelin-A, a endometrial glycoprotein known to block sperm-egg binding in the HZA at low concentrations expresses unusual fucosylated lacdiNAc type glycans. The fucosylated lacdiNAc type sugars have been previously shown to be 15-20 fold more potent ligands of E-selectin. Glycodelin-S a seminal plasma glycoform of …

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 …

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 …

The Evaluation Of Succinylcholine Chloride For Harmful Effects Upon Early-Stage Mouse Embryos, Francis Warren Prescott

Biological Sciences Theses & Dissertations

Succinylcholine (SCh) chloride is a depolarizing neuromuscular blocking agent used to paralyze patients for surgical procedures. Over 1,800 clinical observations of newborn infants have demonstrated that succinylcholine does not cross the placenta in quantities detrimental to fetuses (Kolstad, et al., 1957, Bakhoum, et al., 1957, Hodges, et al., 1959, Duffield, et al., 1958, Dennis, et al., 1954, 1956, McNab, 1955, Coleman, et al., 1956, Lund, 1953). Later investigations which involved comparison of blood levels of SCh in the mother to those in fetuses bolstered the finding that SCh fails to cross the placenta in significant amounts (Moya and Kvisselgaard, 1961, …

Development And Lipid Composition Of The Harpacticoid Copepod Nitocra Spinipes Reared On Different Diets, Gregory M. Weiss, George B. Mcmanus, H. Rodger Harvey

OES Faculty Publications

We reared the harpacticoid copepod Nitocra spinipes on diets of bacteria, a diatom, or a macroalga, evaluating survivorship and growth in short-term (≤ 1 generation) experiments. Lipid content of the copepods and their diets was measured and used as an index of nutrition. Although growth, survivorship and lipid content of N. spinipes were significantly greater when fed the diatom, which had the highest lipid content of the 3 diets, the copepod was able to develop from egg to adult when fed a lipid- poor bacterial diet. Furthermore, this species was able to go through developmental molts without the addition of …

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 …

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. …

Trophic Transfer Of Fatty Acids, Sterols, And A Triterpenoid Alcohol Between Bacteria, A Ciliate, And The Copepod Acartia Tonsa, Melissa C. Ederington, George B. Mcmanus, H. Rodger Harvey

OES Faculty Publications

The incorporation of lipids into the copepod Acartia tonsa and its eggs was measured when it was fed either a bacterivorous ciliate (Pleuronema sp.) or a diatom (Thalassiosira weissflogii). Egg production was 10-fold higher on the diatom diet, whereas hatch success of eggs was the same for algal and ciliate diets. Adult copepods fed diatoms contained more total fatty acid and sterols than copepods fed the ciliate diet, and individual lipids reflected the dietary source. Eggs from diatom-fed copepods had fewer fatty acids but more sterols than eggs from copepods on a ciliate diet. Ciliate-fed copepods and …

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 …

A Kinetic Investigation Of The Mechanism Of Muscle Contraction With A Series Of Nucleotides, Wei Jiang

Theses and Dissertations in Biomedical Sciences

Muscle contraction is thought to be accomplished by sliding of myosin filaments along actin filaments. Although actomyosin naturally uses ATP as an energy source, it can also use other nucleoside triphosphates (NTP) as substrates. In this work, the dependence of rate and equilibrium constants of the various steps in the muscle contraction mechanism upon nucleotide structure was investigated to unravel the dependence of the mechanical properties of the muscle upon changes in different biochemical steps of the mechanism.

The experiments measuring the dissociation of actomyosin by NTP (or mant-NTP) shows that both the rate constant of actomyosin-S1 dissociation and second …

Fish Analysis On Spontaneously Arising Micronuclei In The Icf Syndrome, Michael W. Stacey, M. S. Bennett, M. Hulten

Bioelectrics Publications

The ICF syndrome is a rare disorder where patients show undercondensation of the heterochromatic blocks of chromosomes 1, 9, and 16 along with variable immunodeficiency. The undercondensation of the heterochromatic block appears to be restricted to a portion of PHA stimulated T cells. Patients with this syndrome also show an increase in micronuclei formation. We have used dual colour FISH to investigate the chromosomal content of these micronuclei in PHA stimulated peripheral blood cultures, an EBV transformed B cell line, and also micronuclei observed in vivo from peripheral blood smears. Chromosome 1 appears to be present in a higher proportion …

Cellular Mechanisms Underlying Myogenic Reactivity In Isolated Arterioles, Hui Zou

Theses and Dissertations in Biomedical Sciences

The myogenic reactivity provides one of the principal mechanisms for blood flow autoregulation. The aims of the performed studies described in this dissertation were to test the role of [Ca²⁺]_i and MLC phosphorylation in arteriolar myogenic reactivity and further examine the source(s) of activator Ca²⁺ required to initiate and maintain myogenic vasoconstriction. In addition, the possible underlying mechanism of contractile protein expression was also addressed.

These studies used male Sprague Dawley rats of 200 ~ 350 grams body weight. Experiments were carried out using rat cremaster first order arterioles and mesenteric vessels. Gel electrophoresis and immunoblotting …

Activation Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Rana C. Morris, Bonnie J. Brooks, Panayota Eriotou, Deborah F. Kelly, Sandeep Sagar, K. Lenore Hart, Mark S. Elliot

Chemistry & Biochemistry Faculty Publications

Transfer RNA-guanine ribosyltransferase (TGRase) irreversibly incorporates queuine into the first position in the anticodon of four tRNA isoacceptors. Rat brain protein kinase C (PKC) was shown to stimulate rat liver TG Rase activity, TGRase preparations derived from rat liver have been observed to decrease in activity over time in storage at -20 or -70°C, Contamination of the samples by phosphatases was indicated by a p-nitrophenylphosphate conversion test, The addition of micromolar concentrations of the phosphatase inhibitors sodium pyrophosphate and sodium fluoride into TGRase isolation buffers resulted in a greater return of TGRase activity than without these inhibitors, Inactive TGRase preparations …

In Situ Regulation Of Cytosolic Phospolipase A₂, Beverly A. Rzigalinski

Theses and Dissertations in Biomedical Sciences

The 85 kDa cytosolic phospholipase A₂ (cPLA₂) is an agonist-responsive effector for intracellular signal transduction through the arachidonate cascade. In vitro studies have demonstrated that this enzyme is regulated by sub-micromolar calcium and is specific for arachidonate as the sn-2 fatty acyl group of phospholipid substrates. However, very little data is available regarding in situ mechanisms which govern the activity of cPLA₂. The primarily objective of these studies was to develop an in situ system for the study of cPLA₂, and investigate mobilization of arachidonate during signal transduction events.

Dimethylsulfoxide differentiation of the …

Role Of Heat Shock Protein 70 Kda Cognate In Limiting Thermal Inactivation And Refolding Of Heat-Denatured Nuclear Type I Topoisomerase, Kuo-Kuang Wen

Theses and Dissertations in Biomedical Sciences

Previous studies (Ciavarra et al., 1994) demonstrated that the constitutive 70 kDa heat shock protein (hsc70) protected purified topoisomerase I from thermal injury. In addition, hsc70 was capable of regenerating catalytic activity of heat-denatured topoisomerase I. A whole cell lysate was also active in this reaction assay. The present study demonstrates that heat-denatured topoisomerase I is reactivated by a cytosolic fraction and that this activity is dependent on the presence of cytosolic hsc70. The efficacy of hsc70-mediated refolding of heat-denatured topoisomerase I is greatly enhanced by a cytosolic cofactor(s). In all these refolding reactions, exogenous ATP is not required. Size …

Analysis Of Human Sperm Chromatin Integrity, Silvina M. Bocca

Theses and Dissertations in Biomedical Sciences

Determining potential maternal or paternal sources of abnormal chromosomal constitution gives opportunity for preconception genetic counseling. The most direct determination is achieved by analyzing the nuclear constitution of the gametes.

The present study evaluated the integrity of human spermatozoal nuclear material in the two condensation stages of chromatin and chromosomes. Original semen samples (ORI) and their swim-up fractions (SW, selected for motility) from men of known (donors) and unknown (patients) fertility were analyzed. The extent of chromatin condensation was assessed by light microscopy and flow cytometry during the time course of a chemically-induced decondensation reaction.

Motile spermatozoa were used to …

Translational Regulation Of The C-Jun Proto-Oncogene, Anil Sehgal

Theses and Dissertations in Biomedical Sciences

The v-jun oncogene was originally isolated from the ASV17 virus in 1987. Ever since its isolation, extensive work has been done to understand the role of the v-jun oncogene in cell transformation. The c-Jun protein is a transcription factor which binds to the DNA target TGACTCA. The c-Jun protein binds to DNA in the form of dimers. It can form homodimers with itself and heterodimers with Jun family (JunB and JunD), Fos family (FosB, Fra1 and Fra2), or with CREB family members through the leucine zipper motif. Because the c-jun proto-oncogene plays an important role in cell transformation, extensive work …

The Isolation And Identification Of Polyphenolics From Grape Seeds And Their Activity Towards Specific Cell Lines, Joseph A. Reddy

Chemistry & Biochemistry Theses & Dissertations

Grape seed extract is being marketed in France for cardiovascular use and skin care. The active constituents of both these drugs are certain polyphenols called procyanidins. A similar seed extract has now been obtained from Virginia Chardonnay grape seeds. Ten of the major components in this extract have been isolated in their pure form by the semi-preparative HPLC separation on a YMC ODS-AQ (Octadecylsilane-AQ) column. The ODS-Aq column has been shown to have a better resolving capacity compared to the Spherisorb ODS (Octadecylsilane) column previously described in the literature. This has been attributed to the ODSAQ support which has both …