bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures
Authors: Tuteja Narendra , Phan Tuan-Nghia 
Published: July 2002

Abstract
A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5′-tailed fork was more active than the 3′-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3′ to 5′ along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+&gt;Mn2+&gt;Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (K i approximately 0.85 μm and 2.2 μm, respectively) and ATPase (K i approximately 1.3 μm and 3.0 μm, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.
Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1
Authors: Jones Michelle L. , Woodson William R. 
Published: July 2002

Abstract
We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.
Identification, Separation, and Characterization of Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial β-Oxidation in Higher Plants1
Authors: Bode Kornelia , Hooks Mark A. , Couée Ivan 
Published: July 2002

Abstract
The existence in higher plants of an additional β-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of β-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial β-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial β-oxidation in higher plants.
Gradients in Water Potential and Turgor Pressure along the Translocation Pathway during Grain Filling in Normally Watered and Water-Stressed Wheat Plants
Authors: Fisher Donald B. , Cash-Clark Cora E. 
Published: July 2002

Abstract
The water relations parameters involved in assimilate flow into developing wheat (Triticum aestivum L.) grains were measured at several points from the flag leaf to the endosperm cavity in normally watered (Ψ ≈ −0.3 MPa) and water-stressed plants (Ψ ≈ −2 MPa). These included direct measurement of sieve tube turgor and several independent approaches to the measurement or calculation of water potentials in the peduncle, grain pericarp, and endosperm cavity. Sieve tube turgor measurements, osmotic concentrations, and Ψ measurements using dextran microdrops showed good internal consistency (i.e. Ψ = Ψs + Ψp) from 0 to −4 MPa. In normally watered plants, crease pericarp Ψ and sieve tube turgor were almost 1 MPa lower than in the peduncle. This suggests a high hydraulic resistance in the sieve tubes connecting the two. However, observations concerning exudation rates indicated a low resistance. In water-stressed plants, peduncle Ψ and crease pericarp Ψ were similar. In both treatments, there was a variable, approximately 1-MPa drop in turgor pressure between the grain sieve tubes and vascular parenchyma cells. There was little between-treatment difference in endosperm cavity sucrose or osmotic concentrations or in the crease pericarp sucrose pool size. Our results re-emphasize the importance of the sieve tube unloading step in the control of assimilate import.
Regulation of a Plant SNF1-Related Protein Kinase by Glucose-6-Phosphate
Authors: Toroser Dikran , Plaut Zvi , Huber Steven C. 
Published: July 2002

Abstract
One of the major protein kinases (PKIII) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PKIII activity. Using the continuous assay, along with the fixed-time-point 32P-incorporation assay, we demonstrate that PKIII activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg2+ in the assay from 10 to 2 mm. Under likely physiological conditions (pH 7.0 and 2 mm Mg2+), 10 mm Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PKIII in the following order: Glc-6-P &gt; mannose-6-P, fructose-1,6P2 &gt; ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PKIII activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.
Specific Lipoxygenase Isoforms Accumulate in Distinct Regions of Soybean Pod Walls and Mark a Unique Cell Layer
Authors: Dubbs Wesley E. , Grimes Howard D. 
Published: July 2002

Abstract
Developing seeds constitute a strong sink for the plant and rely on the turnover and mobilization of carbon and nitrogen assimilates to supply the nutrients needed for their maturation. In large part these nutrients emanate from the vegetative organs including leaves and pod walls. Vegetative lipoxygenases (VLXs) accumulate in the paraveinal mesophyll cell layer of soybean (Glycine max L.) leaves where individual isoforms are proposed to play a role(s) as active enzymes or as transient storage proteins. VLXs also are prominent proteins in soybean pod walls, representing approximately 12% of the total soluble protein. Examining the temporal, tissue, and subcellular patterns of individual VLX isoform accumulation and of lipoxygenase activity through pod wall development indicates that VLXD is the principal VLX isoform playing a role in storage in this organ. The major accumulation of VLXD occurs just prior to seed fill within the endocarp middle zone, and protein extracted from this region shows relatively low levels of lipoxygenase activity, suggesting the middle zone may act as a storage tissue. Three other VLX isoforms, VLXA, VLXB, and VLXC colocalize to the cytoplasm of a single discrete cell layer in the mesocarp. Thus, the patterns of VLX cellular and subcellular localization in pod walls suggest independent functions for these different isoforms while also serving as specific markers for a novel cell layer in the pod wall.
Direct Evidence for Requirement of Phosphatidylglycerol in Photosystem II of Photosynthesis
Authors: Hagio Miki , Gombos Zoltán , Várkonyi Zsuzsanna , Masamoto Kazumori , Sato Norihiro , Tsuzuki Mikio , Wada Hajime 
Published: July 2002

Abstract
Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystissp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtainedpgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.
Ultraviolet-B Radiation Impacts Light-Mediated Turnover of the Photosystem II Reaction Center Heterodimer in Arabidopsis Mutants Altered in Phenolic Metabolism
Authors: Booij-James Isabelle S. , Dube Shyam K. , Jansen Marcel A.K. , Edelman Marvin , Mattoo Autar K. 
Published: July 2002

Abstract
Ultraviolet-B (UV-B) radiation can have a negative impact on the growth and development of plants. Plants tolerant to UV-B alleviate these effects using UV-screening pigments that reduce the penetration of UV-B into mesophyll tissue. Little is known about the relative contribution of specific phenolic compounds to the screening capacity of leaves. The D1 and D2 proteins constituting the photosystem (PS) II reaction center heterodimer are targets of UV-B radiation and can be used as an in situ sensor for UV penetration into photosynthetic tissue. Degradation of these proteins occurs under very low fluences of UV-B, and is strongly accelerated in the presence of visible light. Using the D1-D2 degradation assay, we characterized UV-B sensitivity of Arabidopsis mutants (tt4, tt5, andfah1) that are genetically altered in their composition of phenolic compounds. We found that changes in phenol metabolism result in altered rates of PSII reaction center heterodimer degradation under mixtures of photosynthetically active radiation and UV-B. A comparison of D2 degradation kinetics showed increased UV sensitivity of the Landsberg (Landsberg erecta) tt5mutant relative to the Landsberg tt4 mutant and the Landsberg wild type. Despite a lack of flavonoid accumulation, thett4 mutant is not particularly UV sensitive. However, the tolerance of this mutant to UV-B may reflect the increased accumulation of sinapate esters that strongly absorb in the UV range, and may thus protect the plant against environmentally relevant UV-B radiation. This sinapate-mediated protection is less obvious for thett4 mutant of Columbia ecotype, indicating that the relative contribution of particular phenolics to the total screening capacity varies with the genetic background. The role of sinapate esters in UV screening is further substantiated by the results with thefah1 mutant where absence of most of the sinapate esters results in a significantly accelerated degradation of D2 under mixed light conditions. Because the latter mutant is not expected to be deficient in flavonoids, the relative contribution of flavonoids as protectants of PSII reaction center heterodimer against UV-B damage in Arabidopsis needs to be re-evaluated vis-a-vis screening by simple phenolics like sinapate esters.
Transgenic Arabidopsis Plants with Decreased Activity of Fructose-6-Phosphate,2-Kinase/Fructose-2,6-Bisphosphatase Have Altered Carbon Partitioning
Authors: Draborg Henriette , Villadsen Dorthe , Nielsen Tom Hamborg 
Published: July 2002

Abstract
The role of fructose-2,6-bisphosphate (Fru-2,6-P2) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P2 in the leaves. The partitioning of carbon was studied by 14CO2 labeling of photosynthetic products. Plant lines with Fru-2,6-P2 levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of 14C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P2 compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P2 compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P2 in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P2 affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P2 does not contribute significantly to regulation of metabolite levels in darkness.
Major Alterations of the Regulation of Root NO3
− Uptake Are Associated with the Mutation of Nrt2.1 and Nrt2.2 Genes in Arabidopsis
Authors: Cerezo Miguel , Tillard Pascal , Filleur Sophie , Muños Stéphane , Daniel-Vedele Françoise , Gojon Alain 
Published: July 2002

Abstract
The role of AtNrt2.1 and AtNrt2.2genes, encoding putative NO3 − transporters in Arabidopsis, in the regulation of high-affinity NO3 − uptake has been investigated in theatnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220–224) demonstrated that root NO3 − uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO3 −, indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO3 − uptake in this genotype is likely to be due to the constitutive HATS. Root15NO3 − influx in theatnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO3 −availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO3 − uptake in NO3 −-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH4 + in the nutrient solution fails to inhibit 15NO3 − influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH4 +uptake systems in the mutant. These results collectively indicate thatAtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO3 − uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.
Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase
Authors: Noonan Ryan C. , Carter Charles W. , Bagdassarian Carey K. 
Published: July 2002

AbstractAnalysis of the crystal structures for cytidine deaminase complexed with substrate analog 3‐deazacytidine, transition‐state analog zebularine 3,4‐hydrate, and product uridine establishes significant changes in the magnitude of atomic‐scale fluctuations along the (approximate) reaction coordinate of this enzyme. Differences in fluctuations between the substrate analog complex, transition‐state analog complex, and product complex are monitored via changes in corresponding crystallographic temperature factors. Previously, we reported that active‐site conformational disorder is substantially reduced in the transition‐state complex relative to the two ground‐state complexes. Here, this result is statistically corroborated by crystallographic data for fluorinated zebularine 3,4‐hydrate, a second transition‐state analog, and by multiple regression analysis. Multiple regression explains 70% of the total temperature factor variation through a predictive model for the average B‐value of an amino acid as a function of the catalytic state of the enzyme (substrate, transition state, product) and five other physical and structural descriptors. Furthermore, correlations of atomic fluctuation magnitudes throughout the body of each complex are quantified through an auto‐correlation function. The transition‐state analog complex shows the greatest correlations between temperature factor magnitudes for spatially separated atoms, underscoring the strong ability of this reaction‐coordinate species to “organize” enzymatic fluctuations. The catalytic significance for decreased atomic‐scale motions in the transition state is discussed. A thermodynamic argument indicates that the significant decreases in local enzymatic conformational entropy at the transition state result in enhanced energetic stabilization there.
Characterization of Ad5 E3–14.7K, an adenoviral inhibitor of apoptosis: Structure, oligomeric state, and metal binding
Authors: Kim Hee‐Jung , Foster Mark P. 
Published: July 2002

AbstractThe adenovirus E3–14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF‐α and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3–14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein‐protein interaction assays, we demonstrate that the C‐terminal two‐thirds of the protein, predicted to be composed of five β‐strands and one α‐helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high‐order oligomeric state (nonamer) in solution.
Abstract: Surface analytical studies using ion scattering spectrometry, Auger electron spectroscopy and secondary ion mass spectrometry
Authors: Heiland W. , Taglauer E. 
Published: July 2002

Ion scattering spectrometry (ISS), LEED and Auger electron spectroscopy (AES) are combined in one apparatus. By this means the ISS signal can be calibrated as a function of surface composition. This is done for the case of sorption of O and S on Ni. 1,2 By assuming appropriate cross sections an estimate of relative neutralization yields is possible. For the scattering of Ne+ from sulfur adsorbed on nickel the neutralization seems to be a factor of 7 lower than in the case of Ni so that the surface sensitivity is very good (Fig.1). From this figure a detection limit of 10−4 of a monolayer of S on Ni is estimated.
In the case of oxygen adsorption of Ag (110) no simple relations between oxygen signals detected by AES and ISS and the observed LEED pattern have been established. Both signals increase in proportion to the change of the work function with increasing exposure, whereas the Ag signal from ISS decreases (Fig.2). But in the exposure range where (3×1) to (2×1) LEED patterns are observed the oxygen concentration according to AES and ISS does not change from 0.3 to 0.5 of a monolayer. A naive interpretation of the Ag−ISS signal by shadowing may not be justified in that case, even though the detection of oxygen by ISS is a strong indication that at least part of the oxygen is adsorbed above the silver atoms. 4
For many problems ISS can be improved by using an electrostatic anlyzer and a quadrupole mass filter in series, 5 especially when a high yield of true secondary ions buries the peaks of backscattered ions (Figs.3 and 4). In the case of the Nb−Cu sample the high ion yield is due to oxygen contamination of the Nb as shown by the He+ backscattering spectrum, whereas in the Cu−Be case it is obviously due to BeOH contamination as shown by the secondary mass spectrum. This is recorded by using the electrostatic analyzer as an energy window, the secondary ions being accelerated to the appropriate energy. Figure Captions Fig. 1. Auger electron spectroscopy and ion scattering from a S−covered Ni surface. The S concentration amounts to about 0.02 of monolayer. From the spectrum with low impact angle (Ψ = 10,0°) and low scattering angle the sensitivity is estimated to 10−4 of a monolayer. Fig. 2. Auger electron spectroscopy and ion scattering from a O−covered Ag surface. The work function change Δ φ is reported by Engelhard et al.. 3 Fig. 3. Ion scattering from a Nb−Cu surface contaminated by O. The sputtered ions are suppressed by using a mass filter in series with an electrostatic analyzer. Fig. 4. Same technique as used for Fig. 3 in case of a Cu−Be sample.