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 Degree Discipline: Biochemistry
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Functional Characterization of Mtnip/latd’s Biochemical and Biological Function

Functional Characterization of Mtnip/latd’s Biochemical and Biological Function

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Date: December 2013
Creator: Bagchi, Rammyani
Description: Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of ...
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Functional Characterization of Plant Fatty Acid Amide Hydrolases

Functional Characterization of Plant Fatty Acid Amide Hydrolases

Date: December 2010
Creator: Kim, Sang-Chul
Description: Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active ...
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Gene Expression Profiling of the nip Mutant in Medicago truncatula

Gene Expression Profiling of the nip Mutant in Medicago truncatula

Date: August 2007
Creator: McKethan, Brandon Lee
Description: The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number ...
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Genetic Modification of Fatty Acid Profiles in Cotton

Genetic Modification of Fatty Acid Profiles in Cotton

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Date: August 2005
Creator: Rommel, Amy A.
Description: The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.
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Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit

Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit

Date: May 2007
Creator: Coffee Castro-Zena, Pilar G.
Description: A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.
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Identification and Characterization of an Arabidopsis Thaliana Mutant with Tolerance to N-lauroylethanolamime

Identification and Characterization of an Arabidopsis Thaliana Mutant with Tolerance to N-lauroylethanolamime

Date: December 2015
Creator: Adhikari, Bikash
Description: N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of ...
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Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Date: May 2004
Creator: Wanjie, Sylvia W.
Description: The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
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Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver

Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver

Date: May 1992
Creator: Loflin, Paul T. (Paul Tracey)
Description: Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.
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Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

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Date: May 2012
Creator: Meckfessel, Matthew Harold
Description: The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial ...
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
Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings

Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings

Date: August 2010
Creator: Cotter, Matthew Q.
Description: N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. ...
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Kinetic and Chemical Mechanism of 6-phosphogluconate Dehydrogenase from Candida Utilis

Kinetic and Chemical Mechanism of 6-phosphogluconate Dehydrogenase from Candida Utilis

Date: May 1993
Creator: Berdis, Anthony J. (Anthony Joseph)
Description: A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.
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Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium

Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium

Date: August 1993
Creator: Tai, Chia-Hui
Description: Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its ...
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Kinetic and Chemical Mechanism of Pyrophosphate-Dependent Phosphofructokinase

Kinetic and Chemical Mechanism of Pyrophosphate-Dependent Phosphofructokinase

Date: December 1988
Creator: Cho, Yong Kweon
Description: Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of ...
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Luminescence Resonance Energy Transfer-Based Modeling of Troponin in the Presence of Myosin and Troponin/Tropomyosin Defining Myosin Binding Target Zones in the Reconstituted Thin Filament

Luminescence Resonance Energy Transfer-Based Modeling of Troponin in the Presence of Myosin and Troponin/Tropomyosin Defining Myosin Binding Target Zones in the Reconstituted Thin Filament

Date: May 2009
Creator: Patel, Dipesh A.
Description: Mechanistic details on the regulation of striated muscle contraction still need to be determined, particularly the specific structural locations of the elements comprising the thick and thin filaments. Of special interest is the location of the regulatory component, troponin, on the actin filament and how its presence influences the behavior of myosin binding to the thin filament. In the present study: (1) Luminescence resonance energy transfer was used to monitor potential conformational changes in the reconstituted thin filament between the C-terminal region of troponin T and myosin subfragment 1; (2) Location of troponin in previously derived atomic models of the acto-myosin complex was mapped to visualize specific contacts; and (3) Shortened tropomyosin was engineered and protein binding and ATPase assays were performed to study the effect of myosin binding close to the troponin complex. Analysis of the results suggest the following: (1) Irrespective of calcium levels, the C-terminal region of troponin T is located close to myosin loop 3 and a few actin helices that may perturb strong acto-myosin interactions responsible for force production. (2) Atomic models indicate myosin subfragment 1 cannot attain the post- powerstroke state due to the full motion of the lever arm being sterically hindered by ...
Contributing Partner: UNT Libraries
Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Date: August 2013
Creator: Dasgupta, Kasturi
Description: Phloem vascular tissues transport sugars synthesized by photosynthesis in mature leaves by a process called phloem loading in source tissues and unloading in sink tissues. Phloem loading in source leaves is catalyzed by Suc/H+ symporters (SUTs) which are energized by proton motive force. In Arabidopsis the principal and perhaps exclusive SUT catalyzing phloem loading is AtSUC2. In mutant plants harboring a T-DNA insertion in each of the functional SUT-family members, only Atsuc2 mutants demonstrate overtly debilitated phloem transport. Analysis of a mutant allele (Atsuc2-4) of AtSUC2 with a T-DNA insertion in the second intron showed severely stunted phenotype similar to previously analyzed Atsuc2 null alleles. However unlike previous alleles Atsuc2-4 produced viable seeds. Analysis of phloem specific promoters showed that promoter expression was regulated by Suc concentration. Unlike AtSUC2p, heterologous promoter CoYMVp was not repressed under high Suc conc. Further analysis was conducted using CoYMVp to test the capacity of diverse clades in SUT-gene family for transferring Suc in planta in Atsuc2 - / - mutant background. AtSUC1 and ZmSUT1 from maize complemented Atsuc2 mutant plants to the highest level compared to all other transporters. Over-expression of the above SUTs in phloem showed enhanced Suc loading and transport, but against ...
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Manipulations of Sucrose/proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass

Manipulations of Sucrose/proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass

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Date: May 2015
Creator: Khadilkar, Aswad S
Description: Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. ...
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Mechanism of the Adenosine 3',5'-Monophosphate Dependent Protein Kinase

Mechanism of the Adenosine 3',5'-Monophosphate Dependent Protein Kinase

Date: May 1988
Creator: Kong, Cheng-Te
Description: Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher ...
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Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Date: August 2010
Creator: Cao, Te
Description: Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered ...
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Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.

Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.

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Date: May 2005
Creator: McHugh, John
Description: N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the ...
Contributing Partner: UNT Libraries
Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene

Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene

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Date: December 2013
Creator: Salehin, Mohammad
Description: Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence ...
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NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase from Swine Kidney: Characterization and Kinetic Mechanism

NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase from Swine Kidney: Characterization and Kinetic Mechanism

Date: December 1979
Creator: Kung-Chao, Diana T.-Y.
Description: Cytoplasmic 15-hydroxyprostaglandin dehydrogenase from swine kidney was purified to specific activity of 1.2 U per mg protein, by chromatographic techniques. Native molecular weight of enzyme was estimated at 45,000. Enzyme was inhibited by sulfhydryls, diuretics, and various fatty acids. Substrate studies indicated NAD+ specificity and ability to catabolize prostaglandins, except prostaglandin B and thromboxane B. Initial velocity studies gave intersecting plots conforming to a sequential mechanism. 15-keto-prostaglandin exhibited linear noncompetitive production inhibition with respect to either prostaglandin or NAD+; NAD yielded linear competitive production inhibition with respect to NADH. Results, and those of dead-end inhibition and alternated substrate studies, are consistent with an ordered Bi-Bi mechanism: NAD+ is added first, then prostaglandin; then 15-keto-rostaglandin is released, then NADH.
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Noncovalent Crosslinking of SH1 and SH2 to Detect Dynamic Flexibility of the SH1 Helix

Noncovalent Crosslinking of SH1 and SH2 to Detect Dynamic Flexibility of the SH1 Helix

Date: August 2000
Creator: Park, Hyunguk
Description: In this experiment, fluorescent N- (1-pyrenyl) iodoacetamide modified the two reactive thiols, SH1 (Cys 707) and SH2 (Cys 697) on myosin to detect SH1-SH2 a -helix melting. The excimer forming property of pyrene is well suited to monitor the dynamics of the SH1 and SH2 helix melting, since the excimer should only form during the melted state. Decreased anisotropy of the excimer relative to the monomeric pyrene fluorescence is consistent with the disordering of the melted SH1-SH2 region in the atomic model. Furthermore, nucleotide analogs induced changes in the anisotropy of the excimer, suggesting that the nucleotide site modulates the flexibility of SH1-SH2 region.
Contributing Partner: UNT Libraries
NSAID effect on prostanoids in fishes: Prostaglandin E2 levels in bluntnose minnows (Pimephales notatus) exposed to ibuprofen.

NSAID effect on prostanoids in fishes: Prostaglandin E2 levels in bluntnose minnows (Pimephales notatus) exposed to ibuprofen.

Date: August 2009
Creator: Bhandari, Khageshor
Description: Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in ...
Contributing Partner: UNT Libraries