Biochemistry, Biophysics, and Structural Biology Commons^™

The Regulation Of Endothelial And Neuronal Nitric Oxide Synthase Expression In The Preglomerular Vessels By The Increase In Intracellular Calcium Concentration, Stephanie Thomas Montgomery

Chemistry & Biochemistry Theses & Dissertations

This purpose of this study was to examine the regulation of endothelial and neuronal nitric oxide synthase expression in the preglomerular vessels of mature and immature porcine kidneys by the increase in intracellular calcium concentration. Angiotensin II can increase intracellular calcium concentrations in two ways; either through the mobilization of intracellular calcium stores or through the influx of calcium from the extracellular fluid through calcium channels. The aim of this study was to determine the effects of a calcium control, a calcium-free control, L- and T-type voltage-gated calcium channel blockers, an internal calcium store release agent, KCI, and EDTA on …

Biochemical Profiling Of Gout Patients, Tamera Fayre Schlitt

Chemistry & Biochemistry Theses & Dissertations

The purpose of the research is to investigate the biochemical cause of gout in 82 patients. The gouty patients had been diagnosed with gout according to the criterion of the American Rheumatism Association. This gout patient population consisted of 58 men and 24 women with gout whose ages ranged from 11 to 84 years of age with a mean age of 44 years. Key purine metabolism enzyme activity levels and purine metabolite concentrations in the patients' plasma and urine were compared to the mean values of a healthy control group. The control group consisted of 33 males and 25 female …

Development Of Single Nanoparticle Optical Assays For Imaging Single Living Cells, William John Brownlow

Chemistry & Biochemistry Theses & Dissertations

Multi-drug resistance (MDR) has been reported in both prokaryotes and eukaryotes; the pathogenic gram-negative bacteria Pseudomonas aeruginosa can extrude a variety of structurally and functionally diverse substrates via a number of membrane transport systems leading to MDR. We have developed a novel nanoparticle assay to characterize both the membrane transport system composed of the MexAB-OprM efflux pump and the membrane permeability induced by antibiotics. Gold (Au) and silver (Ag) nanoparticles were investigated for use as probes to explore membrane transport in P. aeruginosa.

The surface plasmon absorption (color) of Au nanoparticle solutions was found to change in the presence …

Bacterial Degradation Of Disinfection By-Products In Drinking Water, Laura Inga Fauntleroy

Chemistry & Biochemistry Theses & Dissertations

Chlorine became a major disinfectant for the removal of microbial contaminants in 1914. Current water chlorination procedures yield halogenated disinfection byproducts (DBPs), such as haloalkanes and haloacetic acids (HAAs), due to the reaction of chlorine with naturally occurring organic compounds. Various water utilities have observed decreased HAAs levels in maximum residence time locations (MRTLs), where they were expected to be higher. These MRTLs have low free chlorine residual and high heterotrophic bacteria plate counts. Xanthobacter autotrophicus, GJ-10, is a bacterium that has been shown to contain dehalogenase enzymes and, therefore, can biodegrade HAAs. A number of water-system bacteria were …

Synthesis And Biological Evaluation Of Novel Capacitative Calcium Entry Blockers, Thadigiri Celina Mala

Chemistry & Biochemistry Theses & Dissertations

Studies have shown that 2-aminoethoxydiphenyl borate (2-APB) inhibits thrombin-mediated influx of Ca^{2 +} through store-operated calcium channels in human platelets. Based on this molecule, two sets (boron analogues and the non-boron analogues) were synthesized and characterized by IR, NMR and CHN analysis and evaluated in human platelets to block thrombin-induced [Ca²⁺] elevation.

The synthesis of the non-boron compounds was performed in a simple one step process. According to the literature, some of these compounds were synthesized previously by a complicated two-step process.

All the compounds synthesized were tested for biological activity and the results showed that they …

Real-Time Study Of Multidrug Resistance Mechanism In Pseudomonas Aeruginosa Using Nanoparticle Optics And Single Live Cell Imaging, Sophia Vasou Kyriacou

Chemistry & Biochemistry Theses & Dissertations

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis …

Production Of A Monoclonal Antibody Against Benzo[Α]Pyrene Diol Epoxide Dna Adducts, Brian Peden Austin

Chemistry & Biochemistry Theses & Dissertations

Benzo[α]pyrene is a ubiquitous pollutant produced from the incomplete combustion of organic material such as fossil fuels. It is found in the workplace, urban air, drinking water, and the food supply. Recently, it has been proposed that benzo[α]pyrene may be the causative agent in the formation of lung adenocarcinomas among some Taiwanese women exposed to cooking oil fumes without adequate ventilation. In this study, calf thymus DNA was modified in vitro with benzo[α]pyrene-diol epoxide (BPDE) to a level consistent with that found in biological samples. This DNA of low modification was used as an immunogen in the production of a …

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

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 …

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 …

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 …

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 …

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, …

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 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 …

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 …

Competitive Enzyme Immunoassay Of Catechin In Human Blood Serum, Jay Fedorowicz

Chemistry & Biochemistry Theses & Dissertations

( +)-Catechin is a type of polyphenolic compound, and it is enriched in fruits, plants, and various beverages. Polyphenolic compounds also play an important role in the human diet. Many scientists, especially wine researchers, have proposed that(+)catechin and its polymers could have many positive health effects. Thus, it is essential to learn more about (+)-catechin and other polyphenolics. Presently, high performance liquid chromatography is the only method to detect ( + )-catechin levels in human blood serum, but this method has the high cost of instrumentation and does not provide good sensitivity. The objective of this project is to develop …

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

Chemistry & Biochemistry Theses & Dissertations

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

Structural Characterization Of A Novel Inhibitor Of Hiv Reverse Transcriptase (Hiv Rt), Greggory Jon Woitte

Chemistry & Biochemistry Theses & Dissertations

Human immunodeficiency virus (HIV) infections have become a leading cause of death among young people in the United States today. As the number of HIV infections increases, so too does the cost of treatment. Together, these numbers have prompted an increase in the development of pharmaceutical interventions. HIV reverse transcriptase (HIV RT) has become a suitable target for drug therapy because it is the sole enzyme responsible for HIV replication.

Fucoidan, a sulfated polysaccharide isolated from the brown algae Fucus vesiculosus, has been shown to block a variety of cell adhesion related events including metastasis. In addition, fucoidan has also …

Investigation Of Selenium Status In Hypo-, Hyper- And Euthyroid Children, Ping Sun

Chemistry & Biochemistry Theses & Dissertations

Recently, it has been reported that human type I iodothyronine deiodinase, an enzyme important in the conversion of T₄ to T₃ , is a selenoenzyme. Several studies have reported alterations in plasma selenium level in hypo- and hyperthyroid patients.

Using polarized Zeeman-effect atomic absorption spectroscopy, we measured selenium, zinc, copper and manganese concentrations in the plasma and/or red blood cells in children with or without thyroid disease being seen at the outpatient clinic at Children's Hospital of the King's Daughters. Children with thyroid disease were subdivided into untreated, treated and nonresponsive groups. Data were analyzed using Student's t-test …

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 …

Longitudinal Study Of Plasma And Erythrocyte Selenium Levels In Type I Diabetic Children During Development And Treatment Of Ketoacidosis, Pi-Hsia Fan

Chemistry & Biochemistry Theses & Dissertations

Most studies on the biological activity of selenium have focused on its role at the active site in the ubiquitous enzyme, glutathione peroxidase (GSH-Px) which catalyzes the removal of hydrogen peroxide and organic hydroperoxides (1). A connection between selenium deficiency and a wide range of diseases in animals and humans has been reviewed (2). Development of various forms of human cancer (3), failure of the immune system (4), as well as some cardiovascular diseases (5) have been reported or suspected to correlate with selenium deficiency. However, the physiological function of selenium in these different diseases is not fully understood, and …

Separation And Quantification Of Queuine-Modified And Unmodified Transfer Rna Isoacceptors, Jorge Miguel Pereira De Oliveira Da Silva Santos

Chemistry & Biochemistry Theses & Dissertations

The discovery of a base-exchange modification in the first position of the anticodon of four transfer RNA (tRNA) isoacceptor families has been shown to be correlated with a decrease in the rate of cell division in several tumorigenic tissues. This fact led to the hypothesis that wobbling in the anticodon might be a control point wich mediates the rate of translation. A suitable method for separation of different tRNA isoacceptor families had to be created in order to generate a means for quantitatively measuring degrees of this base modification in tRNA. Radio labelled amino acids were used to charge tRNA …

Structural Characterization Of Fucoidan And Its Role In The Hemizona Assay, Manish S. Patankar

Chemistry & Biochemistry Theses & Dissertations

Fucoidan is a polysaccharide commonly found in brown algae. It is a sulfated polymer of the monosaccharide L-fucose. Fucoidan is thought to function in water retention by algae. Commercially available fucoidan is extracted from a brown algae called Eurus vesiculosus.

This polysaccharide has immense potential as a biologically active agent: (i) it exhibits anticoagulant action, (ii) it has also been reported to block retroviral replication and (iii) it is capable of blocking sperm-egg binding in sea urchin, mouse and rabbit. Recently, fucoidan has also been shown to block the interaction of human sperm and egg in the hemizona assay …

Kinetic Characterization Of A Recombinant C-Terminal Mutant Of Reverse Transcriptase From The Human Immunodeficiency Virus, Thomas S. Heard

Chemistry & Biochemistry Theses & Dissertations

The human immunodeficiency virus (HIV) reverse transcriptase (RT) (EC 2.7.7.49) is the central replication enzyme for HIV. In general, the kinetic mechanism for this and all other polymerases involves the ordered binding of two substrates: a primer-template (PT) followed by a deoxyribonucleoside triphosphate (dNTP). Previous investigations prompted this research when it was discovered that the substrate dNTP, in absence of PT, could protect a recombinant c-terminal mutant HIV-1 RT from inhibition by pyridoxal-5'-monophosphate (PLP), an active-site dNTP inhibitor. In contrast, the non-mutant recombinant HIV-1 RT required both substrates for protection from PLP inhibition. This investigation sought to determine if this …

Biochemical Investigation Of Gout And Its Familial Incidences, Chin-Ling Hsu

Chemistry & Biochemistry Theses & Dissertations

Gout is a chronic metabolic disorder caused by deposition of uric acid on the joint. It is categorized into two types: primary and secondary. Primary gout is uric acid overproduction, caused by excessive synthesis of the purine precursors. Secondary gout occurs also as the result of overproduction or decreased renal excretion of uric acid resulting from other disease processes or medication.

The two purine bases, hypoxanthine and xanthine, liberated from ribosides by the action of nucleoside phosphorylase, are degraded to uric acid as the final excretory product in the purine degradation pathway. Hypoxanthine and xanthine are the physiological substrates of …

Biochemical Analysis Of Human Seminal Plasma, Jamshed Ayub

Chemistry & Biochemistry Theses & Dissertations

Presently there is no single biochemical criterion for differentiating between a potentially fertile male and an infertile male. However, for evaluating male fertility the biochemical analysis of seminal plasma is a very convenient and quantitative technique if it is combined with careful clinical examination.

In seminal plasma analysis, fructose and proteins are chemical indicators of seminal vesicle activity, citric acid and acid phosphatase indicate prostate function while glycerolphosphorylcholine indicates epididymis activity. In this research these five indicators are studied and an attempt is made to establish their normal ranges.

This study shows that the data obtained from the seminal plasma …