Biochemistry Commons^™

Induction Of Apoptosis In Human Prostate Cancer Cells By Resveratrol, Gary Zulfikar Morris

Chemistry & Biochemistry Theses & Dissertations

Recently attention has been brought to trans-resveratrol's {TR) anticancer activity, as determined through a number of cultured cancer cell models. This activity was attributed to TR behaving as an estrogen, and the orientation of TR' s hydroxyl groups. Based on this work it was of interest to determine whether TR would also be toxic in prostate cancer cells; if toxic, did TR induce necrosis or apoptosis in the cells; was it toxic through hormone mediated pathways; and were TR's hydroxyl groups responsible for its biological activity. To this end, cellular viability was assessed in two different prostate cancer cell …

Investigations On The Use Of Ion Mobility Spectrometry For Clinical Chemistry Applications, Henri Parson Patten

Theses and Dissertations in Biomedical Sciences

The major objective of this research is to examine ion mobility spectrometry as a rapid screening tool for specific application to clinical chemistry research and laboratory use. Methodology was developed for target analytes representing several classes of physiologically active substances, including anesthetics, illicit drugs, and their metabolites. The IMS characteristics of animal tissues and other compounds such as amino acids and proteins were determined. Quality assurance and control procedures were developed for specific quality data objectives. Criteria were established relating to use of IMS for assessing the precision and accuracy of data, qualitative screening, and semi-quantitative analyses.

It was found …

Mechanistic Characterization Of The Mitochondrial Type I Dna Topoisomerase And A Study Of Genes Containing Type I Dna Topoisomerase-Related Domains, Jaydee Dones Cabral

Theses and Dissertations in Biomedical Sciences

The mitochondrial type I DNA topoisomerase (mt-topo I) serves an important function in the mitochondrion by relaxing mtDNA supercoils to allow for replication of the mitochondrial genome as well as for gene expression. The mt-topo I's role in essential processes, such as replication and transcription, makes it an ideal candidate as a target for antitumor or antifungal drugs. To gain further insight into mt-topo I mechanism, a cleavage assay and drug inhibition studies were performed. As well, a search for the mt-topo I gene or genes containing type I topoisomerase-related domains was conducted. To characterize the mt-topo I mechanism, the …

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 …

Cytotoxic T Cell Response To Influenza Vaccination In Older Adults, Caroline R. Letter

Chemistry & Biochemistry Theses & Dissertations

Influenza infections result in activation of cellular and humoral immune responses, leading to stimulation of cytotoxic T cells (CTL) and helper T cells with subsequent viral clearance.

By using an assay of granzyme B activity, the in vivo cellular-mediated immune response of old adults was found to be significantly decreased in comparison to young adults. The effect of aging on the CTL response was further investigated via enzyme-linked immunospot technique. A significant increase in number of virus-specific T cells occurred in older adults after vaccination, although the magnitude of the CTL response decreased in comparison to young adults. These data …

Biochemical Composition Of Particles And Dissolved Organic Matter Slong An Estuarine Gradient: Sources And Implications For Dom Reactivity, Antonio Mannino, H. Rodger Harvey

OES Faculty Publications

The chemical composition of high molecular weight dissolved organic matter (DOM) and particulate organic matter (POM) was examined along the salinity gradient of the Delaware Estuary. DOM was collected and fractionated by tangential-flow ultrafiltration into 1-30 kDa (HDOM; high molecular weight) and 30 kDa to 0.2 μm (VHDOM; very high molecular weight) and compared to particles collected in parallel. Polysaccharides comprised 12-43% of particulate organic carbon (POC), 30-56% of VHDOM carbon, and 7.5-19% of HDOM carbon. Hydrolyzable amino acids comprised 17-38% of POC, 5.4-12% of VHDOM carbon, and 1.5-4.2% of HDOM carbon. Only 7-43% of dissolved organic nitrogen in VHDOM …

A Study On The Effects Of The N-Terminal Amino Acid Sequence On The Activation Of Human T-Cell Leukemia Virus Type 1 Protease, Hidayah Muhammad Kendall

Chemistry & Biochemistry Theses & Dissertations

Human T-cell leukemia virus type 1 (HTL V-1) is dependent upon the enzymatic activity of its protease for maturation. Maturation of the protease is facilitated by cleavage of specific amino acid residues, followed by dimerization. The effects of the amino acid sequence located N-terminally to the cleavage site on the ability of the protease to become active were the focus of the current study. These amino acid sequences were contributed by the plasmid vector into which the protease gene was inserted.

Surface probability analyses (SPAs) of the vectors, as well as for native sequences which produce the mature protease and …

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 …

Generation Of Human Chorionic Gonadotropin Beta Subunit Specific Monoclonal Antibody Using A Synthetic Peptide, Zhe Huang

Chemistry & Biochemistry Theses & Dissertations

Human chorionic gonadotropin (hCG) is well known for early immunological and bioassay tests for early detection of pregnancy. Since the hormone specific beta subunit confers unique biological and immunological specificity to hCG, immunochemical measurement of hCG has achieved greater specificity and sensitivity in recent years by using either the beta subunit as an immunogen or a partially reduced beta subunit polypeptide chain. However, antiserum raised against the entire hCG beta subunit usually cross-reacts to some extent with human luteinizing hormone (hLH). hCG has been found to be present in the extract of tumors and minute quantities of hCG are synthesized …

Nitrate Reductase Activity, Nitrate Uptake And Iodine Speciation In The Marine Environment, Chin-Chang Hung

OES Theses and Dissertations

The present method for the determination of new production (NP) by measuring the uptake of added ¹⁵NO₃^– suffers from a number of limitations. In an attempt to improve this situation, this research examined the possibility of estimating ¹⁵NO₃– uptake by measuring the activity of nitrate reductase (NRA). In addition, because it has long been suspected that the biological reduction of IO₃^– to I^– may be mediated by nitrate reductase (NR), this research investigated the ability of NR to catalyze the reduction of iodate to iodide.

An improved method for the determination …

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 …

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 …

Development Of Fluorescent Techniques In The Kinetic Characterization Of Reverse Transcriptase, Kevin Kevin Howerton

Chemistry & Biochemistry Theses & Dissertations

Reverse transcriptase (RT) is an RNA-directed DNA polymerase isolated from human immunodeficiency virus (HIV) and other retroviruses. It has been the primary target for anti-HIV research since the discovery of its vital role in the retroviral life-cycle. The conventional method for carrying out kinetic characterization and inhibition studies of reverse transcriptase involves the use of radioactive materials. Although this method is effective, it is both time consuming, due to a necessary repeated washing step, and hazardous to the worker and environment. Furthermore, strict laws limit the use of radioactive materials to licensed workers and laboratories. In response to these concerns, …

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 …

Developmental And Hormonal Regulation Of The Expression Of Cytosolic Phospholipase A2 Protein Levels In Baboon Placenta, Kathleen Elizabeth Dunstan

Chemistry & Biochemistry Theses & Dissertations

Cytosolic phospholipase A₂ ( cPLA₂) specifically catalyzes the release of arachidonate from membrane phospholipids, thus initiating synthesis of prostaglandins, thromboxanes, and other eicosanoids. The human and nonhuman primate placenta actively synthesize high levels of eicosanoids which play both an autocrine and paracrine role in placental function. Previous studies in Dr. Miriam D. Rosenthal's laboratory have shown that cPLA₂ mRNA levels are developmentally regulated. The present study was designed to determine whether placental cPLA₂ protein levels increased during gestation. Since the marked increases in estrogen levels during normal pregnancy have been shown to enhance morphological and …

The Biogeochemical Cycling Of Dissolved Organic Nitrogen In Estuarine Sediments, David J. Burdige, Shilong Zheng

OES Faculty Publications

Benthic fluxes and pore-water profiles of dissolved organic nitrogen and carbon (DON and DOC, respectively) were determined in seasonal studies at contrasting sites in Chesapeake Bay. Pore-water dissolved organic matter (DOM) concentrations were elevated over bottom-water values, generally increased with depth, and ranged from 15 to similar to 160 μM for DON and ~200-2000 μM for DOC. Pore-water DOM concentrations and the C:N ratio of this material showed spatial (depth) and temporal changes that varied among the sites studied. These trends appeared to be related to differences in the types of sediment organic matter (SOM) undergoing remineralization, as well as …

Iron In Ice Cores From Law Dome, East Antarctica: Implications For Past Deposition Of Aerosol Iron, R. Edwards, Peter N. Sedwick, Vin Morgan, C. F. Boutron, S. Hong

OES Faculty Publications

Total-dissolvable iron has been measured in sections of three ice cores from Law Dome, East Antarctica, and the results used to calculate atmospheric iron deposition over this region during the late Holocene and to provide a preliminary est. of aerosol iron deposition during the Last Glacial Maximum (LGM). Ice-core sections dating from 56-2730 BP (late Holocene) and ∼18 000 BP (LGM) were decontaminated using trace-metal clean techniques, and total-dissolvable iron was determined in the acidified meltwaters by flow-injection analysis. Our results suggest that the atmospheric iron flux onto the Law Dome region has varied significantly over time-scales ranging from seasonal …

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 …

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 …

The Effect Of Growth Hormone Release On Plasma And Erythrocyte Selenium And Zinc Levels In Children, Maria Estela Legal De Reid

Chemistry & Biochemistry Theses & Dissertations

Research studies have suggested that selenium may be important in the release of growth hormone, GH. A selenium-containing amino-acid residue, selenocysteine, has been identified as part of the active site of the three isoenzymes (Type I, II and III iodothyronine 5' -deiodinase) responsible for the synthesis of active thyroid hormone, T_3, which is reported to stimulate GH secretion. The importance of zinc in growth is well- documented because of its involvement in transcription and expression of GH and its intermediary, insulin growth factor I or IGF- I. Thus, selenium and zinc levels in plasma may be altered in response …

Comparison Of The Dichlorination Of Organic N-Chloramines By Caroate And By Bisulfite: A Kinetic Study, Said H. Lubbad

Chemistry & Biochemistry Theses & Dissertations

Bisulfite is commonly used to dechlorinate chlorinated municipal wastewater. However, several studies suggest that bisulfite is not effective in dechlorinating N-chlorinated amino components of wastewater. The purpose of this study was to investigate the effectiveness of an alternate dechlorinating agent, caroate (peroxysulfate), and to compare its rate of dechlorination of three different types of chloramines with that of bisulfite.

Four N-chlorinated amino compounds were investigated: N-chlorodiethylamine, N­ chloroalanylphenylalanine, N-chloroglycylphenylalanine, and N-[2-(N'-chloroimino)­ propanoyl]phenylalanine. These compounds were selected from different families of N­ chloramines to study the effect of structure on dechlorination by both caroate and bisulfite. At pH …

Speciation Of Selenium In Serum, Alice Tsapayi-Musapatike

Chemistry & Biochemistry Theses & Dissertations

We investigated the separation of selenoprotein P and extracellular (serum) glutathione peroxidase using a concanavalin A affinity column. We were able to show that the method does not adequately separate serum glutathione peroxidase from selenoprotein P because human glutathione peroxidase binds strongly to the column. We also found that albumin can interfere with measurement serum of glutathione peroxidase activity when hydrogen peroxide is used as the substrate. When t-butyl hydroperoxide was used as the substrate, less than 10% of the measured activity was due to the presence of albumin. We also found that the pH of the elution buffer plays …

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 …

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 …

Choline Acetyltransferase And Carnitine Acetyltransferase Activity In Human Spermatozoa During Capacitation, Lisa A. Eccles

Theses and Dissertations in Biomedical Sciences

The regional distribution of ChAT activity in human spermatozoa is altered during in vitro capacitation and it correlates with the fertilizing potential of sperm.

Regional immunoreactivity in human spermatozoa as assessed by fluorescent immunocytochemistry was compared with ChAT and CaAT activity determined by enzymatic methodologies. Increasing proportions of sperm exhibited ChAT immunoreactivity along the equatorial region with a concomitant decrease in ChAT reactivity in the midpiece. Also, competitive studies with unlabeled ChAT blocked the equatorial region labeling; the unlabeled CaAT blocked staining along the midpiece region of the tail, suggesting some cross-reactivity of the ChAT antiserum with the CaAT enzyme. …

Application Of Monoclonal Antibodies Against Creatine Kinase Mm And Bb Isoenzymes For Creatine Kinase Mb Isoenzyme Detection, Lei-Chieh Chuang

Chemistry & Biochemistry Theses & Dissertations

Nondiagnostic ECGs and unknown characteristic chest pains increase the diIIiculty of Acute Myocardial Infarction (AMI) diagnosis. Biochemical markers, such as creatine kinase MB isoenzymes (CK-MB) and myoglobin, are employed for the early detection of AMI. Compared to myoglobin, CK-MB reveals a higher specific indicator for AMI detection owing to its abundance in cardiac tissue. Hence, a successful CK-MB detection is expected to improve AMI diagnosis and therapy.

A CK-MB detecting ELISA system was set up based on a sandwich assay principle by using specific CK-B and CK-M monoclonal antibodies. The dimeric nature of the CK-MB isoenzyme provides two epitopes on …

An Enzyme-Linked Immunosorbent Assay For The Detection Of Cardiac Myoglobin Using Monoclonal Antibodies, Deborah F. Kelly

Chemistry & Biochemistry Theses & Dissertations

The most prevalent life-threatening disease worldwide, secondary to coronary artery thrombosis is Acute Myocardial Infarction (AMI). Increases in three clinical markers have been explored as an early means of AMI detection: Cardiac myoglobin, and the two specific cardiac isoforms of Creatine Kinase, CK-MM and CK-MB. However, myoglobin levels have shown to give a more accurate correlation (R = 0.89) than does Creatine Kinase evaluation. Therefore, we have developed a two-site Enzyme-Linked Immunosorbent Assay (ELISA) using monoclonal antibodies against myoglobin isolated from human heart tissue. This assay is based on specific anti-genantibody interactions and is sensitive at the nanogram level. We …