Biochemistry, Biophysics, and Structural Biology Commons^™

Fibronectin-Dependent Activation Of Camk-Ii Promotes Focal Adhesion Turnover By Inducing Tyrosine Dephosphorylation Of Fak And Paxillin, Charles Easley Iv

Theses and Dissertations

Transient elevations in Ca²⁺ have previously been shown to promote focal adhesion disassembly and cell motility. Yet the targets of these Ca²⁺ transients have not been fully examined. In this study, we demonstrate that CaMK-II, a Ca²⁺/calmodulin dependent protein kinase, is activated in response to β1 integrin engagement with fibronectin to influence fibroblast adhesion and motility. We also show that CaMK-II is dynamically localized to the cell surface using Total Internal Reflection Fluorescence microscopy (TIRFm) and that inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and …

The Role Of Secreted Phosphoprotein-24 In Osteoblast Differentiation And Matrix Mineralization, Samuel Ramage

Theses and Dissertations

Secreted Phosphoprotein-24 (Spp24) was initially isolated and characterized as a component of bovine cortical bone matrix. Subsequent characterization has shown it is multiply phosphorylated and homologous to cystatin and TGF-β receptor type II. Spp24 is a minor component of the serum fetuin mineral complex that binds calcium-phosphate minerals and prevents their deposition. The TGF-β receptor homology domain binds BMP-2 weakly in vitro and enhances BMP-2’s osteogenic effects in vivo. The ability of Spp24 to affect BMP activity suggests an important role for Spp24 as a native, bioactive componentof bone that regulates bone development.

Spp24 was highly up-regulated in rat cortical …

Role Of Ceramide-1-Phosphate As A Specific And Potent Activator Of Group Iva Cytosolic Phospholipase A2 Alpha, Preeti Subramanian

Theses and Dissertations

Eicosanoids are potent mediators of inflammatory response whose role has been well established in inflammatory disorders. Release of arachidonic acid by group IVA cytosolic phospholipase A2 α (cPLA2α) is the initial rate limiting step for the production of eicosonoids in response to inflammatory mediators. Previous findings from our laboratory have demonstrated that cPLA2α is directly activated by the emerging bioactive sphingolipid, ceramide-1-phosphate (C1P). In this study, we have developed a modified Triton X-100/phosphatidylcholine (PC) mixed micelle assay which was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the activity of the enzyme increased in …

The Ksga Methyltransferase: Characterization Of A Universally Conserved Protein Involved In Robosome Biogenesis, Heather Colleen O'Farrell

Theses and Dissertations

The KsgA enzymes comprise an ancient family of methyltransferases that are intimately involved in ribosome biogenesis. Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout …

The Role Of Sphingosine Kinase 2 In Cell Growth And Apoptosis, Heidi M. Sankala

Theses and Dissertations

Two isoforms of sphingosine kinase (SphK) catalyze the formation of sphingosine-1-phosphate (SIP). Whereas, SphKl stimulates cell growth and survival, it was found that when overexpressed in mouse NIH 3T3 fibroblasts SphK2 enhances caspase-dependent apoptosis in response to serum deprivation, independently of S1P receptors. Sequence analysis revealed that SphK2 contains a 9 amino acid motif similar to that present in BH3-only proteins. Studies showed that the BH3-only domain, catalytic activity, endoplasmic reticulum (ER) stress, and uptake of calcium by the mitochondria may all contribute to the apoptotic effects of overexpressed SphK2 in NIH 3T3 cells. Further studies in human carcinoma cells …

The Influence Of Adenoviral Infection And The Group Via Calcium-Independent Phospholipase A2 On Hepatic Lipid Metabolism, William Palmer Wilkins Iii

Theses and Dissertations

Sterol regulatory element-binding proteins (SREBP) are transcription factors that regulate genes involved in lipid metabolism especially in the liver. Therefore, hepatic SREBP is significant regulator of systemic lipid metabolism. Evidence demonstrates that insulin and dietary unsaturated fatty acid (UFA) regulate SREBP1 expression and subsequent SREBP1-mediated gene transcription, events that in many instances result in modulation of systemic fatty acid and triglyceride (TG) homeostasis. A series of investigations was designed to uncover novel regulators of SREBP1. Dietary and exogenous addition of UFA has been shown to regulate SREBP function yet, an endogenous source of UFA capable of modulating SREBP remains elusive. …

Atm-Dependent Erk Signaling In Response To Dna Double Strand Breaks, Ashraf Khalil

Theses and Dissertations

Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of …

Phospholipase A2 Induced Monocyte Chemotaxis To Apoptotic Cells, Kwasi Karikari

Theses and Dissertations

Apoptosis is a form of programmed cell death that is essential in such processes as organ and tissue remodeling and maturation of hematopoietic cells. The clearance of apoptotic cells is essential to prevent autoimmune responses to sequestered antigens. This process is mediated by phagocytes of the monocyte lineage. Before phagocytosis can occur, macrophages must be recruited to the apoptotic cells through chemotaxis. Products of the reaction catalyzed by the phospholipases A2 (PLA2) have been shown to induce monocyte chemotaxis either directly or indirectly. Some investigators have implicated a cytosolic calcium-independent PLA2 (iPLA2) in the production of these products during apoptosis. …

Monocyte Modulation Of Disease Pathogenesis And Progression In Localized Aggressive Periodontitis, Chu Ri Shin

Theses and Dissertations

Localized Aggressive Periodontitis (LAgP) is an aggressive, early onset form of periodontitis characterized by a unique myeloid cell phenotype. In addition to its bacterial origin, the unique phenotype of the myeloid cell contributes to disease pathogenesis and progression through mechanisms mediating host inflammatory and immune responses. LAgP monocytes synthesize increased levels of the potent proinflammatory lipid mediator, Prostaglandin E2 (PGE2), preferentially differentiate into dendritic cells, and lead to increased IgG2 production. In addition, levels of Platelet Activating Factor (PAF) have shown to be elevated in the gingival tissue and gingival crevicular fluid of subjects with periodontitis. The aim of this …

Stretch-Dependant Tonic Force Maintenance In Rabbit Epigastric Artery, Krystina Michelle Berg

Theses and Dissertations

The contractile state of vascular smooth muscle (VSM) plays a key role in blood pressure regulation. Abnormal VSM contractility characterizes hypertension and understanding the regulatory mechanisms of VSM may provide some insights to specific treatment of hypertension. Upon muscle stimulation, Ca2+, myosin light chain (MLC) phosphorylation, crossbridge cycling rates and force increase to high levels, but with time, force is maintained while all other parameters of muscle activation fall to low levels. Thus, contraction is divided temporally into early (phasic) and late (tonic) phases, as determined by the underlying regulatory mechanism. Muscles with more phasic characteristics have a higher peak …

Inhibition Of Margination And Diapedesis Of Neutrophils By Protein Synthesis Blockade, Phyllis V. Acquah

Theses and Dissertations

Acute Respiratory Distress Syndrome (ARDS), an age-old clinical problem facing the Emergency Department and Intensive Care Units of all health systems, is a common debilitating lung condition consequent upon severe systemic inflammation. Although several studies have gone into understanding the epidemiology and pathogenesis of the disease thus making way for new advances in treatment strategies, there seems to be no known study tailored to its prevention. Neutrophil extravasation within the tissues during inflammation is the hallmark of this syndrome. Our study sought to block excessive neutrophil infiltration by inhibiting the biosynthesis of some essential proteins necessary for the process. In …

Novel Cancer Therapeutics, The Generation Of Ros, And Cell Survival, Clint Mitchell

Theses and Dissertations

The impact of Ad.mda-7 on the survival of renal cell carcinoma lines (RCC), primary renal epithelial cells, glioblastoma multiforme lines (GBM), and primary rodent astrocytes is unknown. The present studies examine whether the GST fusion protein, GST-MDA-7, and the adenovirus, Ad.mda-7, altered the growth and survival of the A498 and UOK121N RCC lines or radiosensitized GBM, respectively. Due to previous findings that the RCC lines, but not primary renal epithelial cells, were resistant to type 5 adenoviral infection, we used purified GST-MDA-7 protein to show that GST-MDA-7, but not GST, caused a dose-dependent reduction in A498 and UOK121N proliferation but …

Characterization Of The Ftf/Hnf-4 Sites Within The 7alpha- And The 12alpha-Hydroxylase Promoters Involved In The Bile Acid-Mediated Transcription Of Their Regulation, Preeti Pramanik

Theses and Dissertations

Bile acids regulate their own synthesis through a feedback regulatory mechanism of mainly two enzymes in the classic pathway, the 7α-hydroxylase and the 12α-hydroxylase. In the early 1990's it was shown that the regulatory responses of 7α-hydroxylase are mediated at the transcriptional level and since then many positive and negative transcription factors that mediate regulatory response have been identified. An important finding was that the transcription factors regulating the expression of 7α- and 12α-hydroxylase genes are nuclear receptors.One of the first nuclear receptors identified to play a role in the transcription of the 7α-hydroxylase gene was HNF-4 since then many …

Role Of Mdm2 In Cell Growth Regulation, Rebecca Anne Frum

Theses and Dissertations

MDM2 has been shown to induce G0-Gl/S phase arrest. To determine the cell cycle step targeted by MDM2, flow cytometry was employed to detect induction of events during the G1-S phase transition in MDM2-arrested cells. MDM2 overexpression does not prevent expression of cyclin D, cyclin D-CDK mediated phosphorylation of Rb or cyclin E in normal, immortal or tumor-derived cells. However, MDM2 down-regulates cyclin A expression specifically in normal cells, which is associated with G1 arrest. The domain of MDM2 capable of this function is located within its N-terminal 58-109 amino acids. To down-regulate cyclin A, MDM2 requires a functional pl6/Brg …

Characterization Of The Rank Ligand Positive Giant Cell Found In The Interfacial Membrane, Patrick Emerson Jones

Theses and Dissertations

Aseptic osteolysis is a major complication to total joint arthroplasty requiring several thousand people a year to have to undergo revisions of their joint prosthesis. The formation of the interfacial membrane has been associated with aseptic osteolysis leading to the failure of all types of total joints. Recent evidence suggests that RANKL, a potent activator of bone reabsorption, is present in the interfacial membrane. Prior research in this laboratory to determine the source of RANKL in the interfacial membrane has revealed the presence of intense areas of RANKL concentration in the membrane. These areas of RANKL concentration correspond to multiple …

Identification Of The Pla2 Responsible For Prostanoid Synthesis In Response To Inflammatory Cytokines, Chaminda Fernando

Theses and Dissertations

Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory …

Vimentin Overexpression Contributes To The Biological Properties Of Metastatic Head And Neck Cancer Cells, Rachel J. Paccione

Theses and Dissertations

Epithelial to mesenchymal transition occurs in the later stages of epithelial tumor progression, with cells expressing mesenchymal markers. Of these, the intermediate filament protein vimentin is frequently upregulated in metastatic carcinomas. Previously, microarray studies showed that the gene encoding vimentin is highly upregulated in metastatic HN12 cells compared to a related primary tumor cell line. In this study, we confirmed this difference using real-time quantitative PCR, western blot analysis, and immunostaining. Furthermore, EGF and TGF-β, growth factors that induce migration and invasion of HN12 cells, produced synergistic increases in vimentin expression. To assess the contribution of vimentin to the biological …

Modulation Of Gene Expression By Tumor-Derived Mutant P53. Role Of Transactivation In Gain-Of-Function., Mariano J. Scian

Theses and Dissertations

It was hypothesized that the C-terminal sequences for mutant p53 would be required for oligomerization. and oligomerization may be critical for gain-of-function. An N-terminal deletion mutant of p53 that deletes amino acids 1-293 was used as a tool to perform hetero-oligomerization studies. This mutant retains the entire oligomerization domain but dispenses off the transactivation domain and a large portion of the sequence- specific DNA-binding domain. Co-transfection experiments show that p53 del. 1-293 forms hetero-oligomeric complexes with p53-D281G. Also. co-expression of p53 del. 1- 293 with p53-D281G inhibited p53-D28lG-mediated transactivation of the EGFR and MDRl promoters suggesting that hetero-oligomerization inactivates transcriptional …

The Influence Of Lipid Composition On The Binding Of Ldl To Chondroitin 6-Sulphate, Wilma Espiritu

Theses and Dissertations

The interaction between low-density lipoprotein (LDL) and glycosaminoglycans is a key factor in atherosclerosis. The present study examines the characteristics of LDL and its binding properties with the main glycosaminoglycan of the vascular wall, chondroitin 6- sulphate (C6S). The compositional characteristics that were studied for each LDL sample were phase transition temperature, phospholipid content, free cholesterol content, cholesteryl ester content, triglyceride content, and size. Correlations of these characteristics with LDL-C6S binding were analyzed using a turbidity assay. Our results showed that there is no correlation between LDL-C6S binding and phase transition temperature, triglyceride content, or size. Strong correlations were present …

Identification Of The Rna Cis-Elements That Interact With Srp30a To Regulate The Alternative Splicing Of Caspase 9 Pre-Mrna, Prabhat Mukerjee

Theses and Dissertations

Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 …

Cytokine And Chemokine Profiles In A Rat Model Of Hemorrhagic Shock After Immuno-Modulation By Androstenetriol, Kristin E. Paccione

Theses and Dissertations

Further understanding of the cellular and molecular mechanisms involved in traumatic injury, and how they are modulated during drug interaction, can facilitate novel treatment strategies for future trauma patients. We hypothesize that the pharmacological agent, Androstenetriol (AET), up regulates host immune response by modulating the continued expression of mediators, including cytokines. In a double-blinded experiment, rats were hemorrhaged, driven by volume or pressure conditions, then resuscitated with fluids and packed red blood cells following a subcutaneous injection of either vehicle or drug. Blood was collected at various time points and cytokine levels were determined by analyses with both multiplex and …

Characterization Of Rat Pulmonary Carboxylesterase, Timothy J. Wallace

Theses and Dissertations

The 1839 bp cDNA for rat pulmonary carboxylesterase was cloned by reverse transcription polymerase chain reaction (RT PCR) from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to have 99% homology with hepatic CEH. This homology extends to the predicted amino acid sequences which show only six amino acid residue differences in the coding region: three conserved and three nonconserved changes. However, the catalytic activitites of the two proteins are dramatically different. While CEH hydrolyzes cholesterol oleate, the pulmonary …

Chemical Synthesis, Bacterial Expression, And Characterization Of Pro-Gnrh/Gap, A Precursor Protein Of Two Biologically Active Peptide Hormones, Louis E. Mcadory

Theses and Dissertations

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.

GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G⁶LRPGGKR¹³, and correctly cleaves the R^{13 …}

Molecular Cloning And Analysis Of The Promoter For Rat Hepatic Neutral Cholesterol Ester Hydrolase, Ramesh Natarajan

Theses and Dissertations

Neutral cholesterol ester hydrolase (CEH) is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5'-untranslated region as a primer, 1.3 kb of sequence upstream of the ATG initiation codon was amplified and cloned. Primer extension analysis with total RNA from rat primary hepatocytes identified a transcription initiation site, 60 bases upstream from the initiation codon. No typical TATA-box sequences were found upstream from the transcription start site. However, a consensus GC-box, which can bind the positive transcription factor SP1, was found 35 bases upstream from the transcription start site. In addition the …

Space-Based Genetic Cryoconservation Of Endangered Species, Michael Noah Mautner

Chemistry Publications

Genetic materials of endangered species must be maintained, for cryoconservation, permanently near liquid nitrogen temperatures below 77 K. Due to the instability of human institutions, permanent safety is best provided at storage sites that maintain passively the needed low temperatures, and provide barriers to access. The required conditions are available in permanently shaded polar lunar craters with equilibrium temperatures of 8 to 40 K, on the moons of Saturn, and unshielded storage satellites. A genetic depository can be incorporated readily into planned lunar programmes.

Red Blood Cell Stability In Uremic Rats, Cecilia Mermel Maccallum

Theses and Dissertations

Determining the fragility of the red blood cell (RBC) is important for the diagnosis of and evaluation for treatment of several RBC diseases. In part RBC production is controlled through the hormone erythropoietin secreted by the kidney. In a previous study from this laboratory, it was found that RBC were more stable in uremic male rats compared to controls. In this experiment, uremia was induced in four groups of female rats through a two stage nephrectomy. The nephrectomy involved the removal of two-thirds of the left kidney, followed by the removal of the entire right kidney one week later. The …

Regulatory Analysis Of Vimentin Expression In Metastatic Versus Nonmetastatic Breast Cancer Cell Lines, Danielle N. Bird

Theses and Dissertations

The intermediate filament gene family, composed of six classes, shows both tissue and development-specific expression. Vimentin is a unique member of the intermediate filament protein family: although it is expressed in cells of mesenchymal origin, vimentin may also be expressed with other intermediate filament proteins during early stages of development. In some cases, as differentiation continues, vimentin is normally down-regulated whereas other intermediate filament proteins, like desmin, glial fibrillary acidic protein, or neurofilaments are turned on in muscle, glial cells, or neurons, respectively. In some metastatic cancers, including breast and prostate cancers, vimentin is aberrantly expressed, despite the embryonic origin …

Expression, Purification And Characterization Of Hepatitis C Virus Core Protein From E.Coli Using A Chemically Synthesized Gene (1), And Cloning, Expression, Purification And Characterization Of The Major Core Protein (P26) From Equine Infectious Anaemia Virus, Ashley James Birkett

Theses and Dissertations

The hepatitis c virus (HCV) core gene has been chemically synthesized and used to direct the expression of core protein in Escherichia coli (E.coli). When cloned downstream of the inducible T7 promotor the core gene directed the expression of a soluble protein with a molecular weight of 22 kDa. Under native conditions the protein behaved in a manner consistent with the formation of a multimeric structure, which may represent assembled core particles. Further, when examined by electron microscopy a heterogeneous mixture of nucleocapsid-like particles were visible. Core protein was specifically recognized by antibodies present in HCV infected serum, suggesting that …

Cloning, Expression, Purification And Characterization Of Domain B Of Wheat Germ Agglutinin, Ann C. Rice

Theses and Dissertations

Wheat germ agglutinin (WGA) belongs to a family of dimeric chitin binding lectins specific for N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-neuraminic acid (NeuNAc). The polypeptide chain consists of a tandem repeat of four conserved 4.3 kDa domains (A, B, C, D) stabilized by four disulfide bonds. Saccharide binding occurs in the dimer interface where domains of different subunits are in contact (A with D, B with C). An aromatic amino acid rich pocket on one domain (sufficient for saccharide binding) and a polar region on the contacting domain constitute a complete binding site. Saccharide binding affinities may differ among the four unique …

Biochemical Characterization Of The Axolemmal Mitogen For Cultured Schwann Cells, Mark A. Decoster

Theses and Dissertations

The molecule(s) involved at the axon plasma membrane (axolemma) which causes Schwann cells to proliferate has been investigated by three biochemical techniques: 1) Alkaline extraction of axolemma resulted in recovery of 95% of the mitogenic activity and 50% of the protein in the membrane-bound portion of the axolemma. 2) The axonal mitogen for Schwann cells may be associated with heparan sulfate proteoglycans at the axonal surface. Treatment of axolemma with heparitinase, which cleaves the glycosidic bonds of sulfated glycosaminoglycans, allowed mitogenic activity to be solubilized. 3) Treatment of axolemma with heparin, a highly sulfated glycosaminoglycan analogous to heparan sulfate, resulted …