Molecular Biology Commons^™

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 …

Development Of 31p Nuclear Magnetic Resonance Methods For The Study Of Phosphate Metabolisms In E. Coli And B. Subtilis, Fabian M. Dayrit, Emilyn Q. Espiritu, Noreen Gonzalez, Nina Rosario L. Rojas, Jennifer T. Aguilan, Antonio M. Basilio, Edward T. Chainani, Enrico Cruz, Bernadette Matanguihan

Environmental Science Faculty Publications

31P NMR experiments were performed on Escherichia coli and Bacillus subtilis at various temperatures under aerobic and anaerobic conditions. The total soluble intracellular phosphate concentration was estimated to be 2 x 10^-17 mole/cell, while intracellular orthophosphate concentration was around 1 x 10^-17 mole/cell. Addition of glucose resulted in a general decrease in intracellular pH and was accompanied by the formation of sugar monophosphates. The concentrations of soluble intracellular phosphates, both inorganic and organic phosphates, were estimated by integration versus methylene diphosphonic acid (MDPA) standard. Although intracellular and extracellular orthophosphate could be observed, these appear to exchange rapidly on the NMR …

Carmoviruses (Tombusviridae), Feng Qu, Thomas Jack Morris

School of Biological Sciences: Faculty Publications

Carmovirus is one of two officially recognized genera of Tombusviridae. Members of Tombusviridae all have icosahedral virions of about 30 nm in diameter with T = 3 symmetry that consists of 180 coat protein (CP) subunits of about 38- 43 kDa and a single-stranded (ss) RNA genome ranging in size from 4.0 to 4.7 kb. Carmoviruses share recognizable sequence similarity in both the polymerase and structural genes with members of the other genus, Tombusvirus. Sequence comparisons and phylogenetic analysis of these genes support the view that the genera are distinct but closely related. In addition, the genome organization of the …

Construction Of An Escherichia Coli Lac Repressor-Based System To Study Human Cytomegalovirus Gene Expression, Laura Fernanda Cageao-Luchetti

Theses and Dissertations in Biomedical Sciences

An Escherichia coli lac repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the lac repressor was generated (RVlac), and an HCMV-specific promoter was targeted for conditional expression by inserting the lac operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter …

In Vitro Assessment Of The Toxicity Of Cocaine And Its Metabolites In The Human Umbilical Artery, Tessa L. Long

Electronic Theses and Dissertations

An in vitro model was used to assess the effect of cocaine and its metabolites on the umbilical artery. Objectives were to pharmacologically confirm the presence of adrenergic innervation using tyramine, evaluate the ability of cocaine, benzoylecgonine, norcocaine and cocaethylene to potentiate vasoconstriction by serotonin and norepinephrine, examine the ability of ketanserin to block the enhanced vasoconstriction produced by cocaine, and determine displacement of 3 H-ketanserin by serotonin, norepinephrine, tyramine and mianserin. The vasoconstrictive effect of tyramine (100 μM) was enhanced in the presence of cocaine by 257%. Vasoconstrictive effects of serotonin and norepinephrine were significantly enhanced by cocaine by …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

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 …

Expression, Purification, And Characterization Of Nonstructural Protein 2 (Ns2) Of Bluetongue Virus Serotype 17, Hai Lin

Biology

The NS2 protein of bluetongue virus (BTV) serotype 17 (B17) was expressed in large scale as a Glutathione-S-Transferase (GST) fusion protein in E. coli cells. The size of the expressed NS2/GST fusion protein was about 68 kilodaltons (kDa) when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is equal to the sum of the predicted size from the deduced amino acid sequence of the NS2 protein (41 kDa) and the known mass of the GST protein (27 kDa). The NS2/GST fusion protein was purified by absorption to glutathione-conjugated agarose beads (G-beads), and then analyzed on western blots by two …

The Influence Of A Human Repetitive Dna On Genome Stability, Eugenia L. Posey

Electronic Theses and Dissertations

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs …

Fission Yeast Mitotic Regulator Dsk1 Is An Sr Protein-Specific Kinase, Zhaohua Irene Tang, Mitsuhiro Yanagida, Ren-Jang Lin

WM Keck Science Faculty Papers

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known …

Land Use Patterns In Relation To Lake Water Quality In The Great Pond Watershed, Problems In Environmental Science Course (Biology 493), Colby College, Colby Environmental Assessment Team, Colby College

Colby College Watershed Study: Great Pond (2012, 2010, 1998)

Lakes are natural resources, which have many effects on the land surrounding them, They support adjacent communities by providing water and regulating temperatures, helping to define the surrounding ecosystem, and serving as sources of drinking water as well as recreation. The prolonged presence of human activity in a watershed can disturb the physical and chemical cycles of the lake and its surrounding ecosystems (Henderson-Sellers and Markland 1987). Over time, lakes undergo a process called eutrophication, a natural aging process during which the nutrient levels increase and dissolved oxygen levels decrease (Smith and Smith 1998). As the lake ages or becomes …

The Role Of Domain I In Laminin Chain Assembly, George D. Kamphaus

Theses, Dissertations and Capstones

Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu …

Rna Bulge Entropies Correlate With Peptide Binding Strengths For Hiv-1 And Biv Tar Rna Because Of Improved Conformational Access, Brooke Lustig, I Baharand, R L. Jernigan

Faculty Publications, Chemistry

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the ΔΔG values of binding were determined from experimental Kd values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The …

Type I Camp-Dependent Protein Kinase Delays Apoptosis In Human Neutrophils At A Site Upstream Of Caspase-3, Lav K. Parvathenani, E. Stephen Buescher, Enrique Chacon-Cruz, Stephen J. Beebe

Bioelectrics Publications

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had …

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 …

The Electrophysiological Effects Of Acute Thyroid Hormone Exposure On Atrial Myocytes, Jon Paul Fiening

Dissertations

No abstract provided.

Genetic Analysis Of The Nuo Locus That Encodes The Proton Translocating Nadh Dehydrogenase In Escherichia Coli, Holly Jennifer Falk-Krzesinski

Dissertations

No abstract provided.

Identification Of An Amylase-Like Gene In Chironomus Riparius, Loriann Mahurter

Seton Hall University Dissertations and Theses (ETDs)

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Partial Sequencing Of A Balbiani Ring Gene And Development Of A Molecular Probe, Kerri Ann Kurzeja

Seton Hall University Dissertations and Theses (ETDs)

The Environmental Protection Agency is currently developing sediment quality criteria in order to protect the nation's water sources. In order to assist in the development of standards for the disposal of heavy metals, our laboratory is undergoing sediment toxicity studies on Chironomus riparius. It is an EPA approved test organism that spends most of its life in the sediment. Following a previous study (Cochrane et al., 1994), the organism's response to heavy metals will be monitored on the genetic level. Several genes, including a Balbiani ring (BR) gene, which was the focus of this thesis, will be studied to determine …

Sex Chromosome Ratios And Aneuploidy Rates In The Zona Pellucida Selected Spermatozoa From Normal And Subfertile Men, Qinuo Fan Van Dyk

Theses and Dissertations in Biomedical Sciences

The human hemizona has been demonstrated to select spermatozoa with good motility, normal morphology, and the capacity to undergo the zona-induced acrosomal reaction. The studies conducted here are directed at using the human hemizona to investigate two key questions: (1) whether human X-chromosome bearing spermatozoa (X-sperm) and Y-chromosome bearing spermatozoa (Y-sperm) differ in their functional survival time (assessed by their capacity to bind to the human zona pellucida) after prolonged in vitro incubation, and (2) whether hemizona-bound spermatozoa have a reduced aneuploidy rate compared to unbound (and therefore, unselected) spermatozoa?

In the functional survival study, donor spermatozoa were held in …

Interferon Gamma (Ifn-Gamma) Is Necessary For The Genesis Of Acetylcholine Receptor-Induced Clinical Experimental Autoimmune Myasthenia Gravis In Mice., Balaji Balasa, Caishu Deng, Jae Lee, Linda M. Bradley, Dyanna K. Dalton, Premkumar Christadoss, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) …

Ms2 Coat Protein Mutants Which Bind Qβ Rna, Marc Spingola, David Peabody

Biology Department Faculty Works

The coat proteins of the RNA phages MS2 and Qβ are structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qβ. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qβ translational operator led to the isolation of several MS2 mutants that acquired binding activity for Qβ RNA. Some of these also had reduced abilities to …

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 …

Interaction Of Acth And Estradiol In The Regulation Of Growth, Differentiation, And Steroidogenic Maturation Of The Baboon Fetal Adrenal Gland, Maria Gomez Leavitt

Theses and Dissertations in Biomedical Sciences

The present study determined whether the growth of, as well as ACTH receptor and P-450 enzyme messenger ribonucleic acid and/or protein levels in the baboon fetal adrenal are dependent upon fetal pituitary ACTH during mid and late gestation and the mechanism by which placental estrogen modulates ACTH actions. Administration of betamethasone (3mg/day) to baboon mothers on days 60-99 of gestation and to the fetus (0.6 mg/, n = 4) or to the fetus (0.6mg) and mother (6 mg/ml; n = 4) every other day between days 150-164 of gestation (term = 184 days) decreased (P $53β-hydroxysteroid dehydrogenase-isomerase (3β-HSD(+)ctochrome P450 17α-hydroxylase, …

Fluorescence In Situ Hybridization Analysis Of Human Embryos Derived From In Vitro And In Vivo Matured Oocytes, Constance Descisciolo

Theses and Dissertations in Biomedical Sciences

Despite adequate hormonal stimulation, oocytes collected for the purpose of in vitro fertilization and embryo transfer display several levels of nuclear maturity. Preovulatory or mature oocytes, technically those that are Metaphase I or II, are inseminated shortly after aspiration and assessed for fertilization the following day. Prophase I oocytes, also called germinal vesicle-bearing or immature oocytes, require a 24-36 hour period in culture before being exposed to spermatozoa. During this time, the majority of Prophase I oocytes complete nuclear maturation in vitro, progressing from germinal vesicle breakdown through first polar body extrusion. If inseminated, many in vitro matured oocytes fertilize …

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 …

Oral Cancer Screening, Damon Omar Watson

Honors Theses

Cancers of the oral cavity and pharynx have increased throughout the decades. Since oral cancer represents a small percentage of the total cancers in the United States, it is often overlooked. Though this is the case, the results of this cancer can be devastating. To address this issue, an Oral Cancer Screening was organized on August 14, 1996 at the West Michigan Cancer Center in Kalamazoo. This was a joint collaboration between the author, the West Michigan Cancer Center, and the Kalamazoo Valley Dental Society. This event allowed research to take place concerning the data obtained. Confidential results and information …

Local Delivery Of Interleukin 4 By Retrovirus-Transduced T Lymphocytes Ameliorates Experimental Autoimmune Encephalomyelitis., Michael K. Shaw, James B. Lorens, Archana Dhawan, Richard Dalcanto, Harley Y. Tse, Alyssa B. Tran, Colleen Bonpane, Shanti L. Eswaran, Stefan Brocke, Nora Sarvetnick, Lawrence Steinman, Garry P. Nolan, C. Garrison Fathman

Journal Articles: Regenerative Medicine

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.

Studies Of Dictyostelium Discoideum Plasmid Ddp6 And On The Mechanism Of Action Of Rep Proteins From The Ddp2 Plasmid Family, Iman M. Shammat

Biology

Dictyostelium discoideum plasmid Ddp6 from the wild strain NC47.2 is a 5257 bp nuclear plasmid with a copy number of 200-300 per cell. Analysis of the nucleotide sequence and structural organization of the Ddp6 plasmid has identified it as a new member of the Ddp2 plasmid family. Ddp6 contains a single gene that is homologous to the rep gene of Ddp2. As with Ddp2, a single constitutively expressed transcript of 3.3 kb was detected from the rep gene. The Ddp6 Rep open reading frame of 2.8 kb encodes a protein of 932 amino acids. A set of 10 conserved peptide …