The 1.9 Å Structure Of Human Α-N-Acetylgalactosaminidase The Molecular Basis Of Schindler And Kanzaki Diseases,
2010
University of Massachusetts - Amherst
The 1.9 Å Structure Of Human Α-N-Acetylgalactosaminidase The Molecular Basis Of Schindler And Kanzaki Diseases, Nathaniel E. Clark, Scott Garman
Scott Garman
alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human alpha-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human alpha-NAGAL enzyme, we determined three crystallographic complexes with different …
Ethylene Receptors Function As Components Of High-Molecular-Mass Protein Complexes In Arabidopsis,
2010
University of Tennessee - Knoxville
Ethylene Receptors Function As Components Of High-Molecular-Mass Protein Complexes In Arabidopsis, Yi-Feng Chen, Zhiyong Gao, Robert J. Kerriss Iii, Wuyi Wang, Brad M. Binder, G. Eric Schaller
Brad M. Binder
The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4. Methodology/Principal Findings Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of …
Myosin Xi Is Essential For Tip Growth In Physcomitrella Patens,
2010
University of Massachusetts - Amherst
Myosin Xi Is Essential For Tip Growth In Physcomitrella Patens, L Vidali, Gm Burkart, Rc Augustine, E Kerdavid, E Tuzel, M Bezanilla
Magdalena Bezanilla
Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI …
Expression, Purification, And Analysis Of Unknown Translation Factors From Escherichia Coli: A Synthesis Approach,
2010
Western Washington University
Expression, Purification, And Analysis Of Unknown Translation Factors From Escherichia Coli: A Synthesis Approach, Justin D. Walter, Peter Littlefield, Scott P. Delbecq, Gerry Prody, P. Clint Spiegel
Chemistry Faculty and Staff Publications
New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity …
The Role Of Ledgf/P75 In Transcriptional Regulation,
2010
University of Texas at El Paso
The Role Of Ledgf/P75 In Transcriptional Regulation, Jeffrey Ryan Kugelman
Open Access Theses & Dissertations
The Lens Epithelial Derived Growth Factor p75 (LEDGF/p75) is a chromatin bound protein whose cellular function is not yet clearly known. A role in transcriptional regulation had been previously proposed based on its interaction with the basal transcriptional machinery and on its effects on the expression of genes involved in the cellular response to environmental stresses. To further elucidate the function of LEDGF/p75, we conducted a global and unbiased evaluation of the role of this protein in gene expression. To that aim, we performed a microarray analysis of cellular gene expression in cells that are severely depleted of LEDGF/p75. To …
Localization And Functional Analysis Of Plasmodium Falciparum Genes Pfl2550w And Pff0750w,
2010
Loyola University Chicago
Localization And Functional Analysis Of Plasmodium Falciparum Genes Pfl2550w And Pff0750w, Carolyn Jane Strobel
Master's Theses
Malaria is a parasitic disease that causes over a million deaths worldwide each year. Understanding development through the parasite's life cycle is necessary to stop disease transmission. As the genetic basis for the crucial transition from the erythrocytic asexual cycle to gametocytogenesis is unknown, we hope to better understand this transition by studying sexual stage genes and their roles in gametocytogenesis. PFL2550w and PFF0750w are genes upregulated during gametocytogenesis that were identified by a whole-genome microarray comparing gene expression between gametocyte-producing and gametocyte-deficient strains. In this study, PFL2550w was shown to be a soluble protein that is exported from the …
Expression And Characterization Of Antimicrobial Peptides Retrocyclin-101 And Protegrin-1 In Chloroplasts To Control Viral And Bacterial Infections,
2010
University of Central Florida
Expression And Characterization Of Antimicrobial Peptides Retrocyclin-101 And Protegrin-1 In Chloroplasts To Control Viral And Bacterial Infections, Baichuan Li
Electronic Theses and Dissertations
Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration …
Low Cost Production Of Proinsulin In Tobacco And Lettuce Chloroplasts For Injectable Or Oral Delivery Of Functional Insulin And,
2010
University of Central Florida
Low Cost Production Of Proinsulin In Tobacco And Lettuce Chloroplasts For Injectable Or Oral Delivery Of Functional Insulin And, Diane Burberry
Electronic Theses and Dissertations
Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, and C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to …
A Specific Regulatory Role For Sgtα On The Maturation And Activation Of Steroid Hormone Receptors,
2010
University of Texas at El Paso
A Specific Regulatory Role For Sgtα On The Maturation And Activation Of Steroid Hormone Receptors, Atanu Paul
Open Access Theses & Dissertations
Steroid hormone receptors (SHRs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. These receptors regulate various physiological functions in higher ordered eukaryotes. In the absence of hormone these receptors form a complex with molecular chaperones such as Hsp90 and Hsp70 and other cochaperones in the cytoplasm. Association with the Hsp90-Hsp70 chaperone machinery leads to the maturation of the Ligand Binding Domain (LBD) of the receptors and enables the receptors to bind hormone with high affinity. Upon binding with a specific hormone the receptors are translocated into the nucleus where they initiate transcription of specific genes. Although the involvement …
Effects Of The Loss Of Multidrug Resistance Associated Protein 1 On Steroid Homeostasis, Dendritic Cell Function And Compensatory Mechanisms,
2010
University of Texas at El Paso
Effects Of The Loss Of Multidrug Resistance Associated Protein 1 On Steroid Homeostasis, Dendritic Cell Function And Compensatory Mechanisms, Jeffrey Charles Sivils
Open Access Theses & Dissertations
The ATP-binding cassette (ABC) superfamily of membrane transporters use energy derived from ATP to eliminate a variety of exogenous and endogenous compounds from cells including anti cancer and anti viral drugs, metals steroids, bilirubin, cAMP, cGMP, leukotrienes , prostaglandins. The multi drug resistance associated protein family (MRP/ABBC) has been acknowledged as a major player involved in multi drug resistance (MDR), in which cancers stop responding a wide array of structurally and functionally unrelated chemotherapy drugs. Cancer is the second leading cause of death in adults in the United States. Although there have been great strides made the treatment of cancers …
Interactions Of The Cellular Sumoylation System With Influenza A Virus And Its Non-Structural Protein Ns1a (Ns1a),
2010
University of Texas at El Paso
Interactions Of The Cellular Sumoylation System With Influenza A Virus And Its Non-Structural Protein Ns1a (Ns1a), Sangita Pal
Open Access Theses & Dissertations
The most important current anti-influenza weapons, vaccination and antiviral drugs, can be rapidly rendered fully ineffective thanks to the virus's high mutational rate, which produces viruses exhibiting new antigenic properties and structural proteins insensitive to the drug's mechanism of action. One attractive alternative is to develop drugs that modulate the activity of cellular systems either required for viral growth or able to neutralize viral growth. Here we demonstrate that the cellular SUMOylation system, a post-translational modification involving the conjugation of the Small Ubiquitin-like MOdifier (SUMO) to specific protein targets using a Ubiquitin-like enzymatic cascade, interacts closely with influenza virus during …
The Role Of Sumoylation Of Ledgf/P75 In Hiv-1 Infection,
2010
University of Texas at El Paso
The Role Of Sumoylation Of Ledgf/P75 In Hiv-1 Infection, Murilo Tadeu Domingues Bueno
Open Access Theses & Dissertations
The lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional co-activators that protect cells from stresses through modulation of stress and heat shock-related genes. Besides regulating such genes, LEDGF/p75 is also important in the process of HOX gene expression and leukemia transformation driven by the MLL histone methyl transferase complex. By exploiting a similar mechanism of interaction between LEDGF/p75 and MLL, the HIV-1 viral protein Integrase (IN) associates with LEDGF/p75 in order to execute efficient viral DNA integration. This present work has identified that LEDGF proteins are posttranslationally modified by SUMO-1 and -3. SUMOylation was found to target …
Isolation, Characterization And Molecular Cloning Of Dnase Iiib From Drosophila Melanogaster,
2010
University of Texas at El Paso
Isolation, Characterization And Molecular Cloning Of Dnase Iiib From Drosophila Melanogaster, Brenda Cristina Anchondo
Open Access Theses & Dissertations
Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββα-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA non-specific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar …
Ganglioside-Cytokine Interaction In The Induction Of Primary Brain Cell Death,
2010
University of Texas at El Paso
Ganglioside-Cytokine Interaction In The Induction Of Primary Brain Cell Death, John Charles Gorbet
Open Access Theses & Dissertations
Gangliosides have been implicated in multiple pathologies affecting the central nervous system (CNS) and recent research has implicated them in playing an active role in the pathogenesis of multiple sclerosis. Empirical studies and theoretical considerations have suggested the possibility of interactions between gangliosides, like GD3, and pro-inflammatory cytokines present in the nervous system. This study sought to investigate the possibility that either individual gangliosides acting alone or complexed with other species interact with the known immune response factor TNF&alpha to initiate or facilitate cell death in the CNS. I examined the cellular viability and gene expression in primary brain cell …
Phosphorylation Of The Glycine Transporter 1,
2010
University of Texas at El Paso
Phosphorylation Of The Glycine Transporter 1, Javier Vargas Medrano
Open Access Theses & Dissertations
The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the high-affinity glycine transporter 1 (GlyT1) and the clearance of glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the past years, it has been shown that PKC activation diminishes the activity and promoted phosphorylation of several neurotransmitter transporters including the dopamine, serotonin and norepinephrine transporters however, its role is unknown for the glycine transporter. To get insights into the role of PKC activation on GlyT1 regulation, we used three N-terminus GlyT1 isoforms stably expressed in porcine aortic endothelial (PAE) …
Electric Pulses To Prepare Feeder Cells For Sustaining And Culturing Of Undifferentiated Embryonic Stem Cells,
2010
Old Dominion University
Electric Pulses To Prepare Feeder Cells For Sustaining And Culturing Of Undifferentiated Embryonic Stem Cells, Lauren M. Browning, Tao Huang, Xiao-Hong Nancy Xu
Chemistry & Biochemistry Faculty Publications
Current challenges in embryonic-stem-cell (ESC) research include inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gammaradiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed-electric-fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10- nanosecond-electric-pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1s pulse interval, …
Evaluating The Role Of Evolutionarily Conserved Regions Of Ledgf/P75 In Hiv-1 Infection,
2010
University of Texas at El Paso
Evaluating The Role Of Evolutionarily Conserved Regions Of Ledgf/P75 In Hiv-1 Infection, Jose Adrian Garcia
Open Access Theses & Dissertations
LEDGF/p75 is an important cellular co-factor for lentiviral integration. LEDGF/p75-deficient cells are markedly resistant to HIV-1 infection and re-expression of the wild type protein rescues infectivity. Although the molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet known, this co-factor activity requires the interaction of LEDGF/p75 with both the host chromatin and viral integrase. In order to evaluate the involvement of other LEDGF/p75 regions in HIV-1 infection we constructed a panel of deletion mutants targeting clusters of charged residues that are evolutionarily conserved and predicted to be post-translationally modified. These mutants were evaluated for their ability to rescue HIV-1 …
On The Mechanism Of Protein Fold-Switching By A Molecular Sensor,
2010
University of Massachusetts Amherst
On The Mechanism Of Protein Fold-Switching By A Molecular Sensor, Margaret M. Stratton, S N. Loh
Biochemistry & Molecular Biology Department Faculty Publication Series
Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; …
Chloroquine Susceptibility And Reversibility In A Plasmodium Falciparum Genetic Cross,
2010
Old Dominion University
Chloroquine Susceptibility And Reversibility In A Plasmodium Falciparum Genetic Cross, Jigar J. Patel, Drew Thacker, John C. Tan, Perri Pleeter, Lisa Checkley, Joseph M. Gonzales, Bingbing Deng, Paul D. Roepe, Roland A. Cooper, Michael T. Ferdig
Biological Sciences Faculty Publications
Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) are major determinants of verapamil (VP)-reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 x Dd2 cross, we show that the P. falciparum multidrug resistance gene 1 (pfmdr1) interacts with the South-East Asia-derived mutant pfcrt haplotype to modulate CQR levels. A novel chromosome 7 locus is predicted to contribute …
Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression,
2010
Old Dominion University
Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression, W. G. Marshall Jr., B. A. Boone, J. D. Burgos, S. I. Gografe, M. K. Baldwin, M. L. Danielson, M. J. Larson, D. R. Caretto, Y. Cruz, B. Ferraro, L. C. Heller, K. E. Ugen, M. J. Jaroszeski, R. Heller
Bioelectrics Publications
Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly …
