Cell Biology Commons^™

Prostate Specific Membrane Antigen (Psma): Immunoassay Development And Characterization Of Transcriptional Regulation, Zhen Xiao

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCA) is the most common cancer and the second leading cause of death among American men. The high mortality is greatly attributed to the lack of early detection tools and effective treatment for metastasis and relapses. Biomarkers that can discriminate benign from malignant tumor and signal the development of androgen independent and metastatic tumor are needed. A biomarker designated prostate specific membrane antigen (PSMA) has the potential to fulfill this need. The objective of this study is to develop a clinically useful immunoassay for quantitation of serum PSMA and to study the molecular mechanism underlying the upregulation of …

Apoptosis Pathways: Presence And Significance In Ejaculated Human Spermatozoa, Steven Lewis Taylor

Theses and Dissertations in Biomedical Sciences

Ejaculated sperm display markers that are indicative of apoptosis in somatic cells. The question remains as to whether sperm have operative apoptosis mechanisms. The aim of this research was to test the hypothesis that apoptosis markers in sperm and somatic cells are different.

Ejaculated human sperm from patients and donors were separated into high and low motility fractions using Percoll™ gradients. Contaminating cells were removed using anti-CD45 conjugated paramagnetic beads. Fractions were divided into groups: staurosporine, anti-Fas antibody, and hydrogen peroxide treated and control. Direct enzymatic measurement of caspase activity, flow cytometric evaluation of phosphatidylserine translocation, immunoblots, and immunocytochemistry were …

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 …

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 …

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, …

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, …

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 …

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 …

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 …

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 …

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. …

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 …

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 …

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 …

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 …

Analysis Of The Proteoglycan Content In Fresh And Cryopreserved Porcine Cardiovascular Tissues, Yun Hee Shon

Theses and Dissertations in Biomedical Sciences

The purpose of this research was to study the effects of cryopreservation on the proteoglycan (PG) and mineral content of aorta conduit tissue. Proteoglycans from fresh and cryopreserved porcine aorta conduit tissues were isolated by extraction with 4 M guanidine (Gdn) - hydrochloride (HCl) for 48 hours. Quantitative analysis of extracted proteoglycans revealed that the content of proteoglycan material from cryopreserved tissue, measured as uronate and protein per unit weight of wet tissue, was similar to that from fresh tissue. Gel permeation column chromatography studies suggested that proteoglycans present in three CsCl fractions from cryopreserved tissues had molecular weights similar …

Characterization Of A Novel Prostate Tumor-Associated Antigen, Grayson B. Lipford

Theses and Dissertations in Biomedical Sciences

The murine monoclonal antibodies (MAb) TURP-27 and HNK-1 have been shown to detect antigens that are heavily expressed by benign prostatic hyperplasia (BPH) and carcinoma of the prostate (CaP). Western blot analysis of prostate extracts, showed that MAb TURP-27 and MAb HNK-1 bound glycoproteins of 180, 140, 120, 100, 90 and 69 kDa. Studies have shown that the HNK-1 carbohydrate epitope may be involved in cell adhesion and that it is a component of several characterized adhesion proteins. TURP-27 was found to bind at least three of these adhesion proteins: neural cell adhesion molecules (N-CAM), myelin-associated glycoprotein (MAG) and a …

Magnesium Regulation Of Calcium In Essential Hypertension, Elaine Pringle Gordon

Theses and Dissertations in Biomedical Sciences

Essential hypertension is a complex disease which is treated by palliative methods, since the causal factors are as yet unknown. A variety of both genetic and environmental factors probably combine to produce chronic high blood pressure. A genetic model of essential hypertension, spontaneously hypertensive rats (SHR), and its control strain Wistar Kyoto (WKY) were used to investigate a possible environmental causal factor, cellular magnesium deficit, and its impact on the functional ability of the calcium ATPase. Rats were fed either a magnesium-deficient or a magnesium-sufficient diet for 10 weeks, after which the calcium ATPase activity was measured in intact red …

The Effects Of Glutathione On Human Keratinocyte, Manisha N. Trivedi

Theses and Dissertations in Biomedical Sciences

Cultured human keratinocytes were grown in vitro to assess the effects of glutathione, a thiol, on human keratinocyte proliferation. Single keratinocytes were harvested from newborn (greater than 5 days old) foreskin and were grown in culture flasks such that experiments could be performed in approximately 2 to 3 weeks. In order to evaluate the effects of glutathione (GSH) on keratinocyte growth, experiments were performed to determine the growth curve, plating efficiency, and incorporation of radiolabelled thymidine. Growth studies, performed with different concentrations of glutathione, revealed that 0.065 μM was the most effective concentration when compared to control cell cultures, and …

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 …