Biochemistry Commons^™

Experimental And Computational Analysis Of The Synucleins, Agatha Munyanyi

Theses and Dissertations in Biomedical Sciences

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson's disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we …

New Tools For Real-Time Study Of Embryonic Development, Lauren M. Browning

Theses and Dissertations in Biomedical Sciences

Embryonic development represents one of the most complex and dynamic cellular processes in biology, and plays vital roles in understanding of functions of embryonic stem cells (ESCs) and design of ESC-based therapy. Conventional assays and fluorescence-based imaging methods have been widely used for the study of embryonic development. These conventional methods cannot effectively provide spatial and temporal resolutions with sufficient sensitivity and selectivity that are required to depict embryonic development in vivo in real-time at single-cell and single-molecule resolutions. In this dissertation, we have developed a wide range of innovative tools for real-time study of embryonic development. These new tools …

Structural And Functional Study Of Multidrug Membrane Transporters, Feng Ding

Theses and Dissertations in Biomedical Sciences

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for ineffective treatment of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for design of effective therapies. Despite extensive studies over decades, their underlying molecular mechanisms remain largely unknown. In this dissertation, we focus on the study of structures and functions of multidrug membrane transporters, including ATP-binding cassette transporter (BmrA) and Resistance-Nodulation-Cell Division transporter (MexA,B-OprM) in gram-positive and gram-negative bacteria ( Bacillus subtilis and Pseudomonas aeruginosa), respectively. We fused EGFP with N-terminus and C-terminus of …

Design Of In Vivo Assays For Study Of Transport, Biocompatibility And Toxicity Of Nanoparticles, Kerry Jean Lee

Theses and Dissertations in Biomedical Sciences

This dissertation focuses on the design of new in vivo assays for study of transport, biocompatibility and toxicity of nanoparticles (NPs) in zebrafish embryos. We synthesized and purified spherical silver (Ag) NPs with diameters, ranging from 12 to 95 nm, that are stable (non-aggregated) in egg-water media. We developed new imaging approaches to characterize the sizes of single Ag NPs in zebrafish embryos at nanometer resolution by measuring their size-dependent plasmonic spectra and scattering intensity using dark-field optical microscopy and spectroscopy (DFOMS). We used single Ag NPs because they exhibit the high quantum yield (QY) of Rayleigh scattering and resist …

Altering Nitric Oxide Bioavailability And Lipid Profiles In Endothelial Cells By Polycyclic Aromatic Hydrocarbons In Particulate Matter, Liang Yu

Theses and Dissertations in Biomedical Sciences

Epidemiologic studies have demonstrated a significant association between exposure to particulate matter (PM) and atherosclerosis. Polycyclic aromatic hydrocarbons (PAHs) present in particulate matter, are well known to induce oxidative stress and lipid peroxidation via generation of reactive oxygen species (ROS). Lipid peroxidation involves regulating endothelial nitric oxide synthase via inhibition of its activity, and as a result, mediates dilation of coronary arterioles is involved with the pathogenesis of atherosclerosis. However, data on assessment of oxidized lipid formation is limited due to low resolution of mass spectrometer methods. Taking the advantage of a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS) …

Characterization Of Metastasis-Associated Cell Surface Glycoproteins In Prostate Cancer, Lifang Yang

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCa) is a major health problem in males in the United States. Its lethality is mostly attributed to the primary tumor metastasizing to distant sites that are highly resistant to conventional therapies. Serum Prostate Specific Antigen (PSA) is the only protein biomarker used in clinic for prediction of prostate cancer recurrence following local therapies. Nonetheless, PSA lacks the ability to predict the behavior of an individual tumor in an individual patient. Therefore, development of reliable biomarkers for detection of metastatic potential in primary tumors, as well as discovery of new therapeutic targets, is in a great need for …

Analysis Of N-Linked Oligosaccharides Of Prostate-Specific Antigen And Prostatic Acid Phosphatase In Prostatic Fluids, Krista Yaudes White

Theses and Dissertations in Biomedical Sciences

Presently, prostate cancer is the most common cancer afflicting men in the United States, with serum PSA being the "gold standard" protein biomarker used in the clinic for detecting and diagnosing prostate cancer. Nonetheless, serum PSA levels can also be elevated in non-cancerous conditions as well, such as benign prostatic hyperplasia (BPH). Due to this overlap, many unnecessary biopsies and radical prostatectomies occur, leading to patient distress. Despite recent advances to clinical assays which consider other clinical parameters, there is still a great need for improved clinical detection methodologies for prostate cancer, including improved biomarkers. Therefore, this research project aims …

Computational Studies On R67 Dihydrofolate Reductase: An Investigation Into Its Unique Binding Patterns, Chuanyin Shi

Theses and Dissertations in Biomedical Sciences

R67 dihydrofolate reductase (R67 DHFR) is a plasmid encoded enzyme which catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. R67 DHFR is a homo-tetramer and D2 symmetric. It contains only one active site, which spans the central channel of the enzyme. The active site can bind either two reactants (DHF), two cofactors (NADPH) or one of each (NADPH/DHF), which is the productive ternary complex (i.e. the complex which yields product). In order to favor formation of the productive complex, this enzyme exhibits binding cooperativity. Unlike other allosteric enzymes which achieve binding cooperativity through conformational …

Molecular Dynamics Study Of Single Stranded Peptide Nucleic Acids, Anna K. Manukyan

Theses and Dissertations in Biomedical Sciences

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

Structure-Function Studies Of The Camp-Dependent Protein Kinase In Vitro And In Intact Cells, Gary Z. Morris

Theses and Dissertations in Biomedical Sciences

There are 518 protein kinase genes in the human genome; this constitutes about 1.7% of all human genes. The cAMP-dependent protein kinase (PKA) serves as the prototypic model for the study of kinases because it contains a conserved catalytic core shared with all eukaryotic kinases, it is the simplest kinase, and it is one of the best-characterized serine/threonine kinases. PKA is ubiquitous in mammals and regulates multiple physiological mechanisms such as the cell cycle, apoptosis, cell motility, energy metabolism, and gene transcription through a well-defined intracellular signaling pathway. While PKA clearly has a central physiological role it is still unclear …

K+-Induced Smooth Muscle Calcium Sensitization Requires Rhoa Kinase (Rok) Translocation To Caveolae Which Is Inhibited In Non-Neuronal Cell Memory, Nicole Hairrell Urban

Theses and Dissertations in Biomedical Sciences

KC1 causes smooth muscle contraction by elevating intracellular free calcium ([Ca²⁺]i₎, while receptor stimulation activates an additional mechanism termed Ca²⁺- sensitization that can involve activation of ROK and PKC. However, recent studies support the hypothesis that KC1 may also increase Ca²⁺-sensitivity (36). Our data showed that the PKC inhibitor, GF-109203X, did not, while the ROK inhibitor, Y-27632, did inhibit KCl-induced tonic (5’) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited Bay K-8644- and ionomycin-induced MLC phosphorylation and force, but did not inhibit KCl-induced calcium entry or peak …

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 …

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 …

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 …

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 …

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 …

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 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 …

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. …

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 …

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 …

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 …

Determination Of Pancreatic And Salivary Amylase By Enzyme Immunoassay And Their Prevalence In Hyperamylasemic Patients, Sabdra Borgens Ward

Theses and Dissertations in Biomedical Sciences

Currently, amylase determinations are nonspecific for the organ source and are based entirely on the enzymatic properties of amylase to produce a measurable product or byproduct. The determination of pancreatic amylase is important in the diagnosis of acute pancreatitis. Most commercially available tests for amylase employ the measurement of the change in NADH absorbance at 280 nm or of the p-nitrophenol released from a maltotetrose substrate. These are nonspecific measurements of pancreatic amylase and often necessitate other tests to be run such as a serum lipase.

The two predominant isoenzymes of amylase are pancreatic (p-amylase) and salivary (s-amylase); the most …

Structure-Substrate Binding Relationships Of Hiv-1 Reverse Transcriptase, Steve Chien-Wen Huang

Theses and Dissertations in Biomedical Sciences

Human Immunodeficiency Virus, type 1 (HIV-1), is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). HIV-1 reverse transcriptase (RT), a heterodimer p66/p51, has been the major target for treatment of AIDS. The significance of the p51 subunit and the RNase H domain of p66 in terms of their influence on the RNA-dependent DNA synthesis was investigated. Clones of the wildtype HIV-1 RT subunits, p66 and p51, and a recombinant C-terminal deletion mutant, p64, [Barr, P. J. (1987) Bio/Technoloav 5, 486-489] were employed to study the structure-substrate binding relationships of HIV-1 RT. The activity assays of RNA-dependent DNA synthesis on …

Subunit Interactions Of Recombinant Hiv-1 Reverse Transcriptase With Mutations At L289, Jacquelyn R. Smith

Theses and Dissertations in Biomedical Sciences

Reverse transcriptase (RT) is a dimeric enzyme required for replication of the human immunodeficiency virus (HIV). If the subunits of the RT dimer are dissociated, the enzyme is no longer active; therefore, identification of subunit binding sites could lead to potential targets for antiviral therapy. In order to identify where subunit binding of RT occurs, mutations were made at leucine (L) 289, a residue believed to be involved in dimerization through hydrophobic interactions with other leucines. L289 is the central leucine of a leucine repeat sequence which resembles a leucine zipper protein-DNA binding motif. Two mutations, leucine to arginine (L289R) …

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 …

Purification And Characterization Of The Prostate Cancer-Associated Antigen: 7e11-C5, Qi Feng

Theses and Dissertations in Biomedical Sciences

A murine monoclonal antibody (MAb), 7E11-C5, recognizes a prostate organ-specific antigen. Under reducing conditions, the 7E11-C5 antigen exhibited major 100-Kd and minor 70-Kd bands, suggesting that the antigen comprises two peptide chains. The antigen was detected in pooled normal human seminal plasma, xenograft tissue extract of a prostate adenocarcinoma cell line, LNCaP, and tissue extracts of benign prostatic hyperplasia (BPH) and prostate carcinomas, but was not identified in any of the non-prostate tissue extracts tested. Antibodies of the three major prostate-associated antigens (prostatic acid phosphatase (PAP), prostate specific antigen (PSA) and prostate secretory protein (PSP)) were unable to block the …