Life Sciences Commons^™

Isolation And Characterization Of A Genetic Locus That Confers Tolerance To Cadmium And Zinc In Pseudomonas Fluorescens, Szi Fei Feng

Masters Theses

This thesis describes the isolation and characterization of a region containing several genes involved in metal tolerance in the common soil bacterium, Pseudomonas fluorescens ATCC 13525. The DNA region was identified in a cadmium and zinc sensitive mutant, 45-B10, which contains a lacZ-based reporter gene transposon Tn5-B20 inserted in the chromosome. The DNA surrounding the transposon was cloned from this mutant strain and DNA sequencing of this region revealed seven genes that share similarity with genes whose products are involved in the cation-proton antiporter efflux system of other Gram-negative bacteria. Complete DNA sequence was obtained from four of these …

The Role Of Nf-Кb Activation In Breast Carcinoma And Glioblastoma Cell Lines, Kelly T. Murphy

Masters Theses

The nuclear factor-КB (NF-КB)/Rel family of inducible dimeric transcription factors regulates numerous genes associated with the immune, mitogenic, and apoptotic responses. Investigations were made into the role of NF-КB in breast carcinoma (MCF-7, T47D, MDA-MB-231) and glioblastoma (U87MG, U373MG, Ul 18MG) cell lines. NF-КB binding to DNA was quantified using both gel mobility shift assays and transient transfections of a vector containing a luciferase gene under the control of 4 tandem copies of the NF-КB binding sequence. To determine the correlation between NF-КB activation, cellular proliferation, and foci formation, the tumor cells were cultured and analyzed in the presence of …

Effects Of Elevated Carbon Dioxide And Nitrogen Fixation On A Legume-Based System: A Test With Soybean And The Soybean Looper, Pseudoplusia Includens, Angela M. Spickard

Masters Theses

Wild-type and non-nodulated soybean were grown at 367 ± 10 ppm [ambient] and 756 ± 10 ppm [elevated] CO₂ to determine whether nitrogen fixation could minimize the effects of elevated CO₂ on plant nutritional and defensive chemistry. Relative to non-nodulated soybean, wild-type soybean had higher concentrations of leaf nitrogen and proteinase inhibitors, as well as lower C:N ratios and lower phenolics. Elevated CO₂ resulted in decreased leaf nitrogen and proteinase inhibitor contents, and increased C:N ratios and phenolics in both soybean strains. Constitutive levels of phenolics and proteinase inhibitors occurred as predicted by the Carbon/Nutrient Balance hypothesis …

Target-And Stage-Dependent Influences On Axonal Outgrowth Of An Identified Motorneuron In Leech Cns Explants, April Crowley

Masters Theses

The neural circuit that controls rhythmic contraction of the hearts in the medicinal leech, Hirudo medicinalis, provides an amenable model to investigate pathfinding and target selection. The bilaterally paired heart tubes (HT) in leech are eventually innervated by the heart excitor motomeuron (HE) following a period of remodeling that requires the presence of the HT. To begin characterizing possible HT-derived cues, co-cultures of CNS with innervated HT have been used. Previous work demonstrated that young, but not adult, HT was able to support profuse general outgrowth, but the HE extended few projections onto HT of any age. Current studies …

Reactive Oxygen Species Generated By Depletion Of Cellular Glutathione Mediate Cadmium Cytotoxicity, Jun Xu

Masters Theses

Studies were put forth to test whether or not reactive oxygen species, which are influenced by the cellular level of reduced glutathione, mediate the toxicity of cadmium. Cadmium has been a focal point in the health care community in the past few years because it has been shown to induce the development of tumors, interfere with cellular signaling, and cause damage to organs, tissues, and biomolecules. Although various studies and theories have been proposed, little direct evidence, especially from studies utilizing primary cells in culture, has accumulated to show the mechanism by which the cadmium induced toxicity occurs.

Our studies …

Characterization Of Nih-3t3 Mouse Fibroblast Cells Transfected With The Somatostatin Receptor Subtype 2 And The Protein Tyrosine Phosphatase Shp-1, Scott Bradford Cross

Masters Theses

The hormone Somatostatin (SMS) and its analogs have been shown to mediate a variety of cellular actions including inhibition of cellular growth and differentiation of endocrine tumors such as insulinomas and glucagonomas. The mechanisms by which SMS exerts its antitumorigenic effects are poorly understood but are thought to include one of the five Somatostatin receptors (SSTR1-5), and the protein tyrosine phosphatase SHP-1. To determine if the antiproliferative effects of SMS were due to the presence of the SSTR2 receptor or the stimulation of SHP-1, we introduced cDNA's of the SMS receptor SSTR2, SHP-1, and a dominant negative mutant of SHP-1 …

Mechanism By Which Insulin And Its Mimetics, Selenite And Vanadate, Regulate Glucose-6-Phosphate Dehydrogenase Gene Expression, Sanjay M. Jivarj

Masters Theses

Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme of the pentose phosphate pathway. It controls the carbon flow through this pathway, producing reducing equivalents in the form of NADPH to meet the cellular need for reductive biosynthesis and the maintenance of the cellular redox state. Hepatic expression of G6PDH has been shown to be regulated by hormones, nutrients and some growth factors, however the mechanism by which these factors regulate G6PDH gene expression has not been characterized. Here we investigate the mechanism by which insulin and its mimetics, selenate and vanadate, regulate G6PDH gene expression.

Insulin exerts its tissue specific affect …

Stage-Dependent Expression Of Target-Derived Influences Stimulates Axon Outgrowth In Hirudo Medicinalis, Tina M. Harik

Masters Theses

Our lab has been investigating the development of neuronal phenotype and circuitry in Hirudo medicinalis. Early in embryogenesis the heart excitor (HE) motor neuron selectively extends multiple arbors into the body wall prior to heart tube (HT) formation. HE contact with HT primordium at later stages of development results in remodeling such that arbors are elaborated on the HT, while those in the body wall are retracted. HT-derived cues are necessary for HE remodeling (Jellies, 1995. Am. Zool. 35:529; Jellies and Kopp, 1995. Invert. Neurosci. 1:145). HT and body wall explants were co-cultured to investigate the possible influences of …

Characterization Of Swinepox Virus Secretory Polypeptides, Takeshi Shimamura

Masters Theses

Swinepox virus (SPV) is the only known member of the genus Suispoxvirus. SPV causes a mild disease in swine characterized by slight fever, and weight loss, associated with lesions restricted to the skin. There have been few studies on SPV. Other poxviruses, such as vaccinia virus, have been well characterized and the biological activities of their secretory polypeptides have been analyzed. This study describes a major SPV secretory polypeptide with an apparent molecular mass of 35kDa. Poxvirus encoded polypeptides secreted during infection are known to exhibit immunomodulatory activities. The 35kDa SPV secretory polypeptide was tested.for its ability to inhibit …

Effect Of Insulin And Vanadate On Sre-Mediated C-Fos Expression And Jnk Activation, Francisco J. Berguido

Masters Theses

Insulin mimetics are compounds that mimic the action of insulin in the body and are currently being studied as possible therapeutic agents against diabetes. Vanadium has been identified as an element with insulin-like properties with regard to metabolic processes such as lowering blood glucose levels. Few studies have implicated vanadium in mitogenic actions such as cell growth and differentiation. We studied the action of vanadate on the expression of the insulin-induced SRE, a cis-element of the well-characterized mitogenic gene c-fos. We measured this expression by means of a plasmid containing the reporter gene luciferase linked to the sequences from c-fos …

Activation Of Signal Proteins By Insulin And Selenite In Primary Rat Hepatocytes, Ginny Lynn Garlock

Masters Theses

Insulin has a wide range of metabolic and mitogenic effects in cells. The mechanism of insulin action in controlling these events is not well understood, but involves, at least in part, a complex phosphorylation cascade. Selenate has been shown to mimic insulin on several metabolic processes, including the regulation of glucose-6-phosphate dehydrogenase (G6PDH) gene expression. Its mechanism of action, however, is not known. To investigate the mechanisms of insulin and selenate action on the regulation of G6PDH, signal proteins from primary rat hepatocytes were first studied. Insulin and sodium selenate similarly increased tyrosyl phosphorylation of the β-subunit of the insulin …

Isolation And Characterization Of Cadmium Hypertolerant Mutants Of Arabidopsis Thaliana, Santiago Xavier Navarro

Masters Theses

Cadmium is an environmental pollutant which accumulates by deposition from anthropogenic activities. Plants readily take-up cadmium from soil and is concentrated in shoot tissues. Plant and animal systems are affected detrimentally by the exposure to cadmium and are known to produce metal binding peptides as part of detoxification mechanisms. In an attempt to find alternative methods of soil cleanup, efforts are being undertaken to isolate heavy metal accumulating plants. Alternatively, to reduce the accumulation of cadmium in food, plants that exclude heavy metals are also being sought. The objective of this project was to isolate mutants which can be used …

The Role Of Free Radicals In The Plant's Response To Pathogen Attack In Tobacco, Phillip L. Peters

Masters Theses

Previous studies have implicated the involvement of free radicals, specifically reactive oxygen species (ROS), as a key component in plant response to pathogen attack. In this study we examined the effect free radical scavenging compounds have on lesion area, salicylic acid levels, catalase, and superoxide dismutase in response to inoculation with Tobacco Mosaic Virus (TMV), a viral pathogen. Hydroponically grown Nicotiana tabacum (Xanthi nc) were pretreated with one of two antioxidants, either purpurogallin (PPG) or N-acetyl cysteine (NAC). Antioxidants were supplied to the plant via the root system and the leaves were subsequently challenged by inoculation with theTMV. Leaf material …

Expression Of Α4Β1 Integrin On Tissue Eosinophils In The August Rat, John C. Phipps

Masters Theses

The integrin α4β1 is expressed on a wide variety of peripheral leukocytes, and functions in recruitment of these cells to sites of inflammaiton. We infected august (AUG) rats with the helminth parasite Nippostrongylus brasiliensis and later used leukocytes from the peritoneum of the sensitized animals and antibodies against α4β1 integrin to localize that adhesion molecule on the cell surfaces after antigen challenge. We found that after antigen challenge, eosinophils, monocytes, and small lymphocytes, but not mast cells, expressed α4β1 integrin if the animals had been previously infected, but cells from uninfected animals did not show expression of this molecule. Our …

The Effects Of Benzo(A)Pyrene And Dimethylbenzanthracene On Cultured Porcine Aortic Endothelial Function, Michelle Lynn Kagey

Masters Theses

Polycyclic aromatic hydrocarbons are environmental contaminants which have been shown to initiate and promote atherosclerosis and carcinogenesis in various animal species. These findings have been based primarily on the increase in incidence and size of smooth muscle cell tumors in these animals were exposed to benzo(a)pyrene (BaP) or dimethylbenzanthracene (DMBA). The endothelium has also been identified as an important mediator in vascular disease and atherogenesis, although the endothelial response to these PAHs has not been investigated. Thus, the focus of this study was on characterization of the endothelial response to BaP and DMBA. Concentration/response relationships for these chemicals were examined, …

Expression And Regulation Properties Of Pseudomonas L-Arabinose Utilization Plasmids, Mary E. Barecki

Masters Theses

The L-arabinose utilization pathway in Pseudomonas fluorescens is a unique metabolic pathway. In Pseudomonas, L-arabinose is converted to the end product α-ketogluterate and does not involve phosphorylated intermediates. This differs from the well studied L-arabinose pathway found in Escherichia coli which contains phosphorylated intermediates.

The enzymes of the L-arabinose pathway have been shown to be induced in the presence of L-arabinose in P. saccharophila. The current study determined regulatory properties of previously cloned chromosomal fragments of P. fluorescens which confer an L-arabinose positive phenotype to the surrogate host, Pseudomonas putida. Cells of P. putida containing plasmids pPZ221, …

The Flora And Ecology Of Seidman Park, Kent County, Michigan, Pamela J. Laureto

Masters Theses

A floristic study of Seidman Park, Kent County, Michigan was performed. The study identified 521 vascular plants belonging to 96 families and 284 genera. The plant communities of Seidman Park were delimited by identification of dominant species and observation of the physical environment. It was believed that communities existed within the upland forest that could not be identified by observation alone. The use of Non-Metric Multidimensional Scaling (NMS) ordination techniques identified three communities within the upland forest. These were a xeric community, a dry-mesic community and a nutrient-rich community. Three sub-communities of the dry-mesic community were also identified.

The Role Of Nitric Oxide In The Modulation Of The Leukocyte-Endothelial Cell Interactions During Ischemia And Reperfusion, Fernando López-Neblina

Masters Theses

Nitric oxide (NO) is involved in several metabolic pathways and physiological phenomena. The role of NO in inflammation after ischemia and reperfusion (I/R) is controversial. The early phases of inflammation include the leukocyte-endothelial (L/EC) cell interactions which have been divided in rolling, activation, adhesion and migration.

The first experiment was to demonstrate the role of NO in a model of renal I/R. We demonstrated that exogenous NO improves survival and renal function tests after renal I/R, while endogenous NO does not appear to be an important contributor to renal I/R. We also demonstrated that NO modulate the infiltration of neutrophils …

Effects Of Insulin And Insulin-Mimetics On Mrna Levels Of C-Fos And C-Jun In Primary Rat Hepatocytes In Culture, Guangmin Li

Masters Theses

Insulin is capable of regulating cellular and metabolic processes as well as gene expression. Selenate and vanadate are two compounds that have been found to mimic the action of insulin in several tissue and cell types. cFos and cJun are the products of immediate early responsive genes c-fos and c-jun. They are members of the family of the AP-1 (Activator Protein-1) transcription factors. AP-1 transcription factors bind as cJun.cJun homodimer or cJun.cFos heterodimer to the TRE found in the promoter regions of several genes. AP-1 is characterized by its ability to alter gene expression in response to various peptide …

Regulation Of Leukotriene C4 Biosynthesis In Calcium Ionphore A23187-Challeneged August Rat Peritoneal Leukocytes, James M. Huebner

Masters Theses

Leukotriene (LT) C₄ is a potent myotropic product of arachidonic acid 5-lipoxygenation that is thought to play a role in the pathophysiology of immediate hypersensitivity reactions and inflammation. We designed the present study to evaluate the capacity of August (AUG) rat mixed peritoneal leukocytes to generate LTC₄ when challenged with different concentrations of the Ca²⁺ ionophore A23187 and to test the hypothesis that LTC₄ biosynthesis is regulated via a protein kinase C (PKC)-dependent mechanism. The AUG rat, which has a spontaneously high number of peritoneal eosinophils that can be harvested by a simple lavage procedure, is …

Identification Of An Insulin-Responsive Element In The Rat Glucose-6-Phosphate Dehydrogenase Gene, Sheri Lynn Holmen

Masters Theses

The expression of several genes has been shown to be either positively or negatively regulated by insulin at the transcriptional level, however no single insulin responsive DNA element has yet been identified. The transcription factors responsible for stimulation or inhibition of gene expression by insulin also remain to be identified. In recent years, expression of the gene for glucose-6-phosphate dehydrogenase (G6PDH) in primary rat hepatocytes has been found to be influenced by hormones such as insulin, dexamethosone, and glucagon. The molecular mechanism(s) however, underlying hormonal regulation of G6PDH gene transcription remains to be elucidated. The focus of this study was …

Differential Distribution Of D4 And D2 Dopamine Receptors In Rat Brain: An Autoradiographic Study Using [3H]Ym-09151-2 And [3H]Raclopride, Nancy C. Stratman

Masters Theses

Extrapyramidal side effects (EPS) are associated with most antipsychotics for the treatment of schizophrenia since these agents antagonize dopamine D₂ receptors in the nigrostriatal pathway, a brain system that regulates motor functions. Clozapine, an atypical antipsychotic agent in that it does not produce EPS, was found to be more selective for the D₄ than the D₂ receptor. The purpose of this study was to locate the D₄ receptor in rat brain by autoradiographic comparison studies with [³H]YM-09151-2, a D₂ , D₃, and D₄ radioligand, to [³H]raclopride, a D …

Characterization And Expression Of Pseudomonas Fluorescens L-Arabinose Dehydrogenase: Genetic Elements In Pseudomonas Putida, Carol M. Wierenga

Masters Theses

An assay based on the spectrophotometeric properties of NAD/NADH was developed to detect the degradation of L-arabinose to L-arabinolactone. This assay was used to determine the expression of the L-arabinose utilization genes contained in Pseudomonas fluorescens and in the plasmid, designated pPZ221, contained in Pseudomonas putida. The results from this assay, in conjunction with various growth kinetics, indicate that pPZ221 is inducible and repressible for L-arabinose utilization. Thus, the P. fluorescens chromosomal insert in pPZ221 for L-arabinose utilization does appear to contain the regulatory genes necessary for controlling the expression of an L-arabinose operon.

Estrogen Induced Eosinophil Migration Into The August Rat Uterus Is Associated With Intercellular Adhesion Molecule Expression, Daniel R. Orcutt

Masters Theses

Estrogen causes an influx of eosinophils into the uterus in female rats. The diapedesis of eosinophils out of the vasculature has also been observed in allergic reactions, parasitic infections, and atopic dermatitis, and has been associated with cellular adhesion molecules (CAMs). Antibody blocking studies against ICAM-1/ LFA-1 and VCAM-1/VLA-4 have suggested that both ligand/receptor sets of CAMs are involved in the selective recruitment of eosinophils out of the vasculature. ICAM- 1 is expressed on the vascular endothelium and decreases in expression when IL-4 is introduced. VLA-4 is present on eosinophils and is suspected to allow selective migration of eosinophils out …

The Effect Of Insulin Mimetics On Enzymes Of Fatty Acid Biosynthesis, Eric A. Berg

Masters Theses

Fatty acid biosynthesis, an insulin regulated pathway, is the process of producing fatty acid chains from 2 and 3 carbon units. Two enzymes in this pathway that insulin affects are glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS). Sodium vanadate and sodium selenate have demonstrated insulin-like effects in several tissue and cell types. These mimetic effects include increasing glucose transport, regulating glycolytic enzymes, normalizing plasma glucose levels and activities of insulin regulated enzymes in diabetic rats. Our goal was to examine effects of sodium vanadate and sodium selenate on fatty acid biosynthesis in two insulin responsive systems: Isolated rat hepatocytes …

Expression Of Integrated And Methylated Hiv-Ltr In Human T-Cells And Monkey Kidney Cells By Frog Virus 3 Infection, Celene M. Spangler

Masters Theses

DNA methylation has been implicated in the suppression of transcription of a large spectrum of eukaryotic genes. Frog virus 3 (FV3) contains genomic DNA that is the most extensively methylated of all known animal viruses. However, FV3 gene expression is tightly regulated in a sequential fashion in infected cells. Therefore, FV3 must have evolved mechanism(s) to overcome the inhibitory effects of DNA methylation. FV3 has been shown to induce expression of methylated foreign genes in transient transfections. This study was designed to establish if this FV3 induced expression of methylated genes could be demonstrated in stable cell lines which contain …

Complete Nucleotide Sequence Of Psti-L Fragment Of Tanapox Virus Genomic Dna, Sarah J. Neering

Masters Theses

Tanapox virus (TPV), of the genus Yatapoxvirus, is a human poxvirus that was originally isolated from the Tana River Valley in Kenya in 1962. TPV produces a mild disease characterized by a short febrile illness associated with one or more nodular skin lesions. Although there have been limited studies on TPV, other poxviruses such as vaccinia virus, have been extensively characterized and their genomic DNAs sequenced. In order to examine the molecular features of TPV, we have cloned approximately 17% of the genomic DNA. TPV DNA was digested with Pstl restriction endonuclease and cloned into pUC19. The Pst …

Site-Directed Mutagenesis Of The Putative Processing Site Of Recombinant Human Immunodeficiency Virus Reverse Transcriptase, Susan M. Poppe

Masters Theses

The human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). This retrovirus, encodes a RNA dependent DNA polymerase, reverse transcriptase (RT), which is essential to virus replication. Biochemical analysis indicated that RT is composed of 2 polypeptides of 66,000 and 51,000 daltons (p66 and p51) which combine to form a heterodimer. This heterodimer is thought to arise from the post-translational processing where p66 is made and either autocatalytic or proteolytic cleavage events result in a heterogeneous p66/p51. Site-directed mutagenesis was used at the proposed processing site to probe HIV-RT processing.

Three prokaryotic expression vectors …

Cloning And Sequencing Of The Eco Rii Monomer Fragment Of Satellite Dna From The Mouse Mus Musculus, Timothy Patrick Boyle

Masters Theses

Total mouse satellite DNA was isolated in cesium chloride gradients in the presence of the dye Hoechst 33258. This satellite fraction was digested with Bst Nl, an isoschizomer of the restriction enzyme Eco RII, and the 234 base pair monomer fragment was cloned into the plasmid vector pBS. Dideoxynucleotide sequencing of the cloned DNA yielded significant homology with the published consensus sequence of mouse satellite DNA (92%).

Receptor Localization And Distribution On Normal And Diabetic Cells By Electron Microscopy, Vance L. Kincaid

Masters Theses

The objective of this research was to evaluate receptor localization and distribution on normal and diabetic cells by electron microscopic analysis.

To achieve this objective, gold-adsorbed and ferritin-conjugated macromolecules were used to determine the location and concentration of specific receptor binding sites on the cells surfaces. To facilitate the use of these ultra-structural histocytochemical markers, normal and diabetic Chinese hamster and other nondiabetic cells were either grown in vitro on ultra thin plastic films or embedded in Lowicryl K4M and pre- or post-labelled with these marker molecules.

Analysis of the data obtained from electron micrographs of these labelled cells was …