Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2

Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2

Date: August 2013
Creator: Taei, Nasrin
Description: Hypertrophic cardiomyopathy (HCM), a heart-related abnormality, is the most prevalent cause of sudden death in young athletes at sporting events. A cluster of cardiomyopathy mutations are localized in β-cardiac myosin at the N-terminal region of subfragment-2. Using resonance energy transfer probes, a synthetic peptide model system was developed to study stability of the coiled coil (S2 fragment) structure by determining monomer-dimer equilibrium of the peptide. Fluorescence resonance energy transfer and MacroModel software suite were used to obtain distance measurements along with measurement of coiled coil formation. The model peptide was used to characterize the effects of disease-causing-mutations and examine potential candidate drugs (polyamines) to counteract effects of mutations causing HCM. Distance measurements between donor and acceptor probes obtained by computational simulation and fluorescence resonance energy transfer (FRET) were consistent. Measurements also agreed with simulations of unlabeled wildtype, indicating coiled coil structural stability of the peptide. Interaction of the site-specific antibody with the peptide strongly inhibited dimerization and destabilized coiled coil structure of the peptide. Presence of negatively charged glutamate residues in the region of subfragment-2 strongly suggested a potential interaction site for positively charged polyamines. Binding of certain polyamines, such as poly-L-Lysine 11 residues and poly-D-Lysine 17 residues, demonstrated the ...
Contributing Partner: UNT Libraries
Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans.

Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans.

Date: May 2009
Creator: Stewart, Neil
Description: Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint ...
Contributing Partner: UNT Libraries
DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers

DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers

Date: August 1997
Creator: Chiu, Angela Chen-Yen
Description: The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.
Contributing Partner: UNT Libraries
Advanced Molecular and Microbial Techniques: a Complete Laboratory Notebook

Advanced Molecular and Microbial Techniques: a Complete Laboratory Notebook

Date: May 1998
Creator: Brito-Rodriquez, Carmen Lydia
Description: The purpose of this project is to produce a complete and thorough notebook that may be used to supplement laboratory coursework. Its intent is to be used primarily by the students to aid them in understanding background information and the proper laboratory procedures involved in various types of experiments. The laboratory notebook is a summation of all the experiments and procedures used in the six-credit hour Advanced Microbial and Molecular Biology (BIOL 5160) course offered during the summer semester at the University of North Texas. This class is a team taught effort by Professors O'Donovan and Kunz. The course is constructed as an intensive practice exercise to teach the student about gene mutations, biosynthetic pathways, preparation and analysis of plasmid DNA, and many other topics included in the notebook.
Contributing Partner: UNT Libraries
Functional Neural Toxicity and Endocrine Responses in Mice Following Naphthalene Exposure

Functional Neural Toxicity and Endocrine Responses in Mice Following Naphthalene Exposure

Date: August 2010
Creator: Colbert, Crystal
Description: Polycyclic aromatic hydrocarbons (PAHs) are a well studied and diverse class of environmental toxicants. PAHs act via the aryl hydrocarbon receptor (AhR), and studies have suggested that PAHs may elicit neurological and estrogenic effects. Doses of PAHs between 50 to 150 ppm may elicit neurotoxicity in rodent models. The present study investigated the effects of naphthalene on in vivo steroidogenesis in Swiss Webster male mice, and in vitro neural function of Balb-C/ICR mice frontal cortex neurons. These data suggest that naphthalene may not elicit steroidogenic effects at concentrations ranging from 0.2 to 25 mg/kg/day, following a 7 day subcutaneous dosing regime. In addition, naphthalene may cause functional toxicity of frontal cortex neurons at concentrations of 32 to 160 ppm naphthalene.
Contributing Partner: UNT Libraries
Effector response of the aspartate transcarbamoylase from wild type  Pseudomonas putida  and a mutant with 11 amino acids deleted at the N-terminus of PyrB.

Effector response of the aspartate transcarbamoylase from wild type Pseudomonas putida and a mutant with 11 amino acids deleted at the N-terminus of PyrB.

Date: May 2002
Creator: AsFour, Hani
Description: Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.
Contributing Partner: UNT Libraries
Nucleotide Sequence of a Bovine Arginine Transfer RNA Gene

Nucleotide Sequence of a Bovine Arginine Transfer RNA Gene

Date: May 1996
Creator: Eubanks, Aleida C. (Aleida Christine)
Description: A single plaque-pure lambda clone designated λBA84 that hybridized to a ˆ32P-labeled bovine arginine tRNA was isolated from a bovine genomic library harbored in a lambda bacteriophage vector. A 2.3-kilobase segment of this clone was found to contain an arginine transfer RNAccg gene by Southern blot hybridization analysis and dideoxyribonucleotide DNA sequencing. This gene contains the characteristic RNA polymerase III split promoter sequence found in all eukaryotic tRNAs and a potential RNA polymerase III termination site, consisting of four consecutive thymine residues, in the 3'-flanking region. Several possible cis-acting promoter elements were found within the 5'-flanking region of the sequenced gene. The function of these elements, if any, is unknown.
Contributing Partner: UNT Libraries
Subcellular Localization of N-acylphosphatidyl-ethanolamine Synthase in Cotyledons of Cotton Seedlings

Subcellular Localization of N-acylphosphatidyl-ethanolamine Synthase in Cotyledons of Cotton Seedlings

Date: December 1995
Creator: Sriparameswaran, Anuja
Description: N-acylation of phosphatidylethanolamine (PE) with free fatty acids catalyzed by N-acyl phosphatidylethanolamine (NAPE) synthase was reported in cotyledons of 24-h-old cotton seedlings. Here I report subcellular localization of this enzyme. Differential centrifugation, sucrose density gradient fractionation,aqueous two-phase partitioning and electron microscopy techniques were utilized to elucidate subcellular site(s) of NAPE synthase. Marker enzymes were used to locate organelles in subcellular fractions. Differential centrifugation indicated that NAPE synthase is present in more than one organelle and it is a membrane bound enzyme. Sucrose density gradient fractionations indicated that NAPE synthase is present in membranes derived from endoplasmic reticulum (ER),Golgi and possibly plasma membrane (PM) but not mitochondria, glyoxysomes or plastids. Aqueous two-phase partitioning experiments with cotton and spinach tissues supported these results but Goigi appeared to be the major site of NAPE synthesis. Electron microscopy of subcellular fractions was used to examine isolated fractions to provide visual confirmation of our biochemical results. Collectively, these results indicate that NAPE is synthesized in plant ER, Golgi and possibly PM.
Contributing Partner: UNT Libraries
Photoactivatable Quantum Dots in Super-Resolution Microscopy of Muscle

Photoactivatable Quantum Dots in Super-Resolution Microscopy of Muscle

Date: December 2010
Creator: Akel, Amal
Description: Super-resolution 3D imaging was achieved using newly synthesized photoactivatable quantum dot (PAQ dot) probes. Quantum dots were modified with a novel quencher system to make them photoactivatable. The unique properties of these PAQ dots enable single-fluorophore localization in three dimensions using a confocal microscopy optical sectioning method. Myosin and tropomyosin of rabbit myofibrilar bundles were specifically labeled with the newly synthesized PAQ dot. A sufficient number of single quantum dots were photoactivated, localized and reduced to their centroid and then reconstructed to a super-resolution image. The acquired super-resolution image shows a lateral and an axial sub-diffraction resolution and demonstrates ultrafine striations with widths less than 70 nm that are not evident by conventional confocal microscopy. The striations appear to be related to nebulin thin filament binding protein. This newly developed imaging system is cutting edge for its high resolution and localization as well its simplicity and convenience.
Contributing Partner: UNT Libraries
Subcloning and Nucleotide Sequence of the xylO/PUWCMA Region from the Pseudomonas putida TOL Plasmid pDK1

Subcloning and Nucleotide Sequence of the xylO/PUWCMA Region from the Pseudomonas putida TOL Plasmid pDK1

Date: December 1997
Creator: Guigneaux, Michelle M. (Michelle Marie)
Description: The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.
Contributing Partner: UNT Libraries
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