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High resolution fractionation, characterization, and studies of ADP-ribose polymers

Description: A method for the high resolution fractionation of ADP-ribose polymers has been developed that allows isolation of highly purified polymers of defined size and branching fequency. The key features of the method are purification using dihydroxyboronyl-BioRex 70 and fractionation by anion exchange HPLC.
Date: August 1992
Creator: Kiehlbauch, Charles C. (Charles Coffey)
Partner: UNT Libraries

Synthesis, purification and characterization of small mono(ADP-ribosyl)ated molecules in the ADP-ribose elongation reaction catalyzed by poly(ADP-ribose)polymerase

Description: The ADP-ribose elongation catalyzed by poly(ADP-ribose) polymerase (PARP) [EC 2.2.2.30] has been partially characterized utilizing mono (ADP-ribosyl)ated polyamines. Arginine methyl ester (AME)-(ADP-ribose) and agmatine (AGMT)-(ADP-ribose) were synthesized enzymatically with a eukarytic mono(ADP-ribosyl) transferase and cholera toxin, respectively.
Date: December 1993
Creator: Pacheco-Rodriguez, Gustavo
Partner: UNT Libraries

Studies on ADP-Ribose Polymer Metabolism in Cultured Mammalian Cells Following DNA Damage

Description: ADP-ribose polymer metabolism has been studied in human cells derived from a patient with Glutamyl Ribose Phosphate Storage Disease (GRPSD) and in mouse C3H1OT1/2 cells following oxidative stress induced by hydrogen peroxide (H202 ). It has been postulated that GRPSD resulted from an abnormality in ADP-ribose polymer metabolism. This study has shown that these cells exhibit reduced poly(ADP ribose) polymerase activity which is proposed to result from modification of the enzyme with ribose phosphate groups. The modification in the polymerase is proposed to be secondary to a defect in either ADP-ribosyl proteinlyase or an overproduction of a cellular phosphodiesterase. The metabolism of ADP-ribose polymers was rapidly altered by H202 and there were independent effects on adenine nucleotide pools. The results suggest that ADP-ribose polymer metabolism is involved in cellular defenses to oxidative stress.
Date: May 1991
Creator: Maharaj, Geeta
Partner: UNT Libraries

Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver

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.
Date: May 1992
Creator: Loflin, Paul T. (Paul Tracey)
Partner: UNT Libraries

Phosphorus Turnover and Photosynthesis

Description: The participation of phosphorus in biological oxidation-reduction reactions of the type found in glycolysis ADP + PO{sub 4}H{sup -} + 3-phosphoglyceraldehyde + DPN{sup +} = 3-phosphoglycerate{sup -} + 2H{sup +} + DPNH + ATP has suggested theories in which similar reactions are proposed for photosynthesis. In these theories the reducing power of photosynthesis is utilized not only for reduction of carbon dioxide but also, by means of coupled oxidations, for the generation of high-energy phosphate bonds, or in the last reference directly for the generation of high-energy phosphate. Since in these theories acyl phosphate is formed from inorganic phosphate, they are amenable to proof without isolation of particular intermediates, by means of radioactive phosphorus. It would be expected that the rate of conversion of inorganic phosphate to organic phosphate would be greater in light than in the dark. They have investigated this possibility under a variety of conditions and are unable to substantiate the theories.
Date: November 1, 1947
Creator: Aronoff, Sam & Calvin, Melvin
Partner: UNT Libraries Government Documents Department

The Beginning of Kinesin's Force-Generating Cycle Visualized at 9Angstrom Resolution

Description: We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in ''ejecting'' adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the ''neck linker''). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.
Date: June 20, 2007
Creator: Sindelar, Charles V. & Downing, Kenneth H.
Partner: UNT Libraries Government Documents Department

Studies on Poly (ADP-ribose) Synthesis in Lymphocytes of Systemic Lupus Erythematosus Patients

Description: A method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product.
Date: December 1991
Creator: Chen, Hai-Ying
Partner: UNT Libraries

Poly(ADP-ribose) Synthesis as a Function of Growth and DNA Fragmentation

Description: This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.
Date: December 1981
Creator: Levi, Viktorya
Partner: UNT Libraries

Structure Function Relationships of ADP-Glucose Pyrophosphorylase and Branching Enzyme: Manipulation of Their Genes for Alteration of Starch Quanlity and Quantity

Description: Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit can be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. ...
Date: February 16, 2006
Creator: Preiss, Jack
Partner: UNT Libraries Government Documents Department

[Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

Description: Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.
Date: April 1, 1999
Creator: Okita, T.W.
Partner: UNT Libraries Government Documents Department

Quantification of Poly(ADP-ribose) in Normal and in DNA-Damaged Cells

Description: This work presents the development of a new highly sensitive and selective chemical assay for poly(ADP-ribose) which is routinely useful for the determination of polymer levels in vivo. This method was used to carefully measure poly(ADP-ribose) levels in normal and in DNA-damaged cells. The results of these studies strongly suggest that synthesis of poly(ADP-ribose) is involved in some aspect of DNA repair. A review of the literature is presented in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in aspects of transcription, in DNA syn thesis, and in DNA repair largely based on evidence from in vitro studies. It is apparent that current methodology has not allowed the routine quantification of poly(ADP-ribose) in vivo, hence the lack of i^n vivo data concerning the function(s) of the polymer. The body of this work presents the development of two chemical methods for the quantification of poly(ADP-ribose) and the application of one of these methods to the measurement of polymer levels in normal and DNA-damaged cells. Preliminary studies are presented on the utilization of combined gas chromatography/mass spectroscopy for the selective quantification of nucleoside derivatives. A second method makes use of the unique chemistry of the polymer for quantification. The polymer was selectively adsorbed to dihydroxyboryl-sepharose which allowed the removal of most RNA, DNA, and protein from the samples. The polymer was hydrolyzed to the unique nucleoside 2'—^-l*'-ribosyladenosine by digestion with venom phosphodiesterase and bacterial alkaline phosphatase. The 1-N^-etheno derivative of ribosyladenosine was formed by reaction with chloroacetaldehyde and this derivative was seperated from other fluorescent species by reversed phase high pressure liquid chromatography.
Date: December 1980
Creator: Sims, James L.
Partner: UNT Libraries

[Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990]

Description: The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of the gene that encodes for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. During the last two years we have obtained information on the structure of this enzyme from both potato tuber and rice endosperm, using a combination of biochemical and molecular biological approaches. Moreover, we present evidence that this enzyme may be localized at discrete regions of the starch grain within the amyloplast, and plays a role in controlling overall starch biosynthesis in potato tubers.
Date: December 31, 1990
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase genes. Progress report, [April 15, 1990--April 14, 1991]

Description: The long term goal of this project is to assess the feasibility of increasing the conversion of photosynthate a key regulatory enzyme in starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is primarily regulated by the gene activation, expression, and allosteric regulation of ADPglucose pyrophosphorylase, as well as starch synthase, and branching enzyme. During the last year we have elucidated the structure of both subunits which compose this tetrameric enzyme and determined the temporal and spatial expression of the genes encoding each subunit as well as their correlation to starch biosynthesis. Genomic clones to both subunits have also been isolated and the gene structure of the small subunit determined. Transgenic potato plants have been produced containing deletions of the small subunit promoter. Currently, cis acting elements and their involvement in spatial and temporal expression are under investigation.
Date: December 31, 1990
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1988--April 14, 1989]

Description: During this period researchers have been successful in determining the structure of the rice pyrophosphorylase gene. Potato tuber ADPglucose pyrophosphorylse purification and structure studies were carried out as well as recombinant DNA studies. Evidence suggests that the tuber form is made up of subunits with similar molecular weights and immunological relatedness. In contrast, the spinach leaf enzyme and presumably the maize endosperm species is composed of two dissimilar sununits encoded by different genes.
Date: December 31, 1989
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

[Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Summary of progress, [April 15, 1991--April 14, 1992]

Description: The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of genes encoding enzymes that may be rate-limiting in starch biosynthesis. In developing storage tissues such as tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. In view of the regulatory role of ADPglucose pyrophosphorylase in starch biosynthesis at both the genetic and biochemical level, we have focused our attention on the genes that encode for this enzyme in potato tubers. The proposed objectives of the grant were to (1) analyze the structure of the tuber enzyme, (2) isolate and characterize the structure of its genes, and (3) identify the regulatory elements controlling ADPglucose pyrophosphorylase during plant development. During the last two and 1/2 years we have met or have made considerable progress in achieving these objectives as discussed in more detail below.
Date: December 31, 1992
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

[Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, April 1993--January 1994

Description: Part I of this research concerns patterns of gene expression of ADPG-PP in native and transgenic potato plants. The expression of both potato ADPG-PP subunits were analyzed on the transcript and antigen levels. The small and large subunits were coordinately expressed during tuber development suggesting a role for the temporal regulation of ADPG-PP expression as well as providing further support for earlier work in the heterotetrameric subunit structure of the tuber enzyme. Part II involves studies on the structure-function relationships of ADPG-PP, more specifically the mutagenesis of the large and small subunit DNAs of ADPG-PP.
Date: April 1, 1994
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1987--April 14, 1988]

Description: Many agronomically important crops are viewed as significant resources of renewable energy. Overall crop productivity could be increased if the efficiency of photoassimilate conversion into dry matter such as starch were improved in storage tissues. Starch production is controlled by the catalytic activity of ADPglucose pyrophosphorylase in the first step of starch biosynthesis. This research focuses on the genetic structure and molecular mechanisms by which it is controlled during plant development and how it is affected by environmental and hormonal conditions. The current goal is to isolate the genes for this enzyme present in both cereal endosperm and potato tuber tissues, and to elucidate its structure and the controlling sequences responsible for gene expression. The long term goal is the improvement of starch production in storage organs by manipulating this gene so that it encodes an enzyme refractive to inorganic phosphate inhibition.
Date: December 31, 1988
Creator: Okita, T. W.
Partner: UNT Libraries Government Documents Department

Energetic control of intact chloroplast photosynthesis

Description: The induction phase of photosynthesis was studied in intact chloroplasts isolated from spinach. Within 15 sec of illumination, stromal ADP is phosphorylated to give an ATP/ADP ratio of 1.4. A transthylakoid pH gradient of about 4.1 units developed during the first min, and was associated with a low note of linear electron flow from H/sub 2/O to NADP/sup +/. In the following 2 to 3 min, both the pH gradient and ATP/ADP ratio declined to new steady state levels (3.9 pH units and a ratio of 0.9), whereas O/sub 2/ evolution rose rapidly to 110 ..mu..mol/mg chlorophyll-h. Prolonging the induction phase by omission of catalase or by addition of 2 mM phosphate resulted in a high pH gradient and ATP/ADP ratio. Ribose 5-phosphate is known to reverse partial inhibition resulting from catalase omission or high phosphate, hence its effect on the pH gradient and ATP/ADP ratio was investigated. Ribose 5-phosphate was found to lower both these parameters under conditions chosen to restrict turnover of the carbon cycle. This result with ribose 5-phosphate underscores the importance of its product, ribulose 5-phosphate and the carboxylation product, 3-phosphoglycerate, in serving as sinks for ATP and thereby preventing inhibition of electron flow by an excessive pH gradient.
Date: unknown
Creator: Slovacek, R E & Hind, G
Partner: UNT Libraries Government Documents Department

Ocean thermal energy conversion preliminary data report for the November 1977 GOTEC-02 cruise to the Gulf of Mexico Mobile Site

Description: This is the second in a series of preliminary data reports from cruises to potential Ocean Thermal Energy Conversion (OTEC) sites in the Gulf of Mexico. The data are from the GOTEC-02 cruise to a site at approximately 29/sup 0/N, 88/sup 0/W, the Mobile Site. Twelve oceanographic stations were visited. Due to bad weather, the results are scanty. The reader will note that much of the data is questionable. Current meter results are presented elsewhere (Molinari, Hazelworth and Ortman, 1979). Determinations of the biomass indicators - chlorophyll a, phaeophytins and adenosine triphosphate - and zooplankton, are presented. Results were generally those that might have been predicted from previous studies in the area.
Date: March 1, 1980
Partner: UNT Libraries Government Documents Department