bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the Binding and Inhibition Sites in the Calmodulin Molecule for Ophiobolin A by Site-Directed Mutagenesis
Authors: Kong Au Tai , Chow Leung Pak 
Published: July 2002

Abstract
Ophiobolin A, a fungal toxin that affects maize and rice, has previously been shown to inhibit calmodulin by reacting with the lysine (Lys) residues in the calmodulin. In the present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin molecule by site-directed mutagenesis, either by deleting them or by changing them to glutamine or arginine. We found that each of these three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding site and is responsible for all of the inhibition of ophiobolin A. When Lys-75 was removed, Lys-77 could then react with ophiobolin A to produce inhibition. Lys-148 was shown to be a binding site but not an inhibition site. The Lys-75 mutants were partially resistant to ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues were mutated, the resulting calmodulins were very resistant to ophiobolin A. Furthermore, Lys residues added in positions 86 and/or 143 (which are highly conserved in plant calmodulins) did not react with ophiobolin A. None of the mutations seemed to affect the properties of calmodulin. These results show that ophiobolin A reacts quite specifically with calmodulin.
Developmental and Environmental Effects on the Expression of the C3-C4 Intermediate Phenotype inMoricandia arvensis
Authors: Rylott Elizabeth L. , Metzlaff Karin , Rawsthorne Stephen 
Published: July 2002

Abstract
Cellular anatomy and expression of glycine decarboxylase (GDC) protein were studied during leaf development of the C3-C4intermediate species Moricandia arvensis. Leaf anatomy was initially C3-like and the number and profile area of mitochondria in the bundle-sheath cells were the same as those in adjacent mesophyll cells. Between a leaf length of 6 and 12 mm there was a bundle-sheath-specific, 4-fold increase in the number of mitochondrial profiles, followed by a doubling of their individual profile areas as the leaves expanded further. Subunits of GDC were present in whole-leaf extracts before the anatomical development of bundle-sheath cells. Whereas the GDC H-protein content of leaves increased steadily throughout development, the increase in GDC P-protein was synchronous with the development of mitochondria in the bundle sheath. The P-protein was confined to bundle-sheath mitochondria throughout leaf development, and its content in individual mitochondria increased before the anatomical development of the bundle sheath. Anatomical and biochemical attributes of the C3-C4 character were present in the cotyledons and sepals but not in other photosynthetic organs/tissues. In leaves and cotyledons that developed in the dark, the expression of the P-protein and the organellar development were reduced but the bundle-sheath cell specificity was retained.
In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts1
Authors: Wischmann Bente , Hamborg Nielsen Tom , Lindberg Møller Birger 
Published: July 2002

Abstract
Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.
Developmental Expression of Violaxanthin De-Epoxidase in Leaves of Tobacco Growing under High and Low Light
Authors: Bugos Robert C. , Chang Sue-Hwei , Yamamoto Harry Y. 
Published: July 2002

Abstract
Violaxanthin de-epoxidase (VDE) is a lumen-localized enzyme that catalyzes the de-epoxidation of violaxanthin in the thylakoid membrane upon formation of a transthylakoid pH gradient. We investigated the developmental expression of VDE in leaves of mature tobacco (Nicotiana tabacum) plants grown under high-light conditions (in the field) and low-light conditions (in a growth chamber). The difference in light conditions was evident by the increased pool size (violaxanthin + antheraxanthin + zeaxanthin, VAZ) throughout leaf development in field-grown plants. VDE activity based on chlorophyll or leaf area was low in the youngest leaves, with the levels increasing with increasing leaf age in both high- and low-light-grown plants. However, in high-light-grown plants, the younger leaves in early leaf expansion showed a more rapid increase in VDE activity and maintained higher levels of VDE transcript in more leaves, indicating that high light may induce greater levels of VDE. VDE transcript levels decreased substantially in leaves of mid-leaf expansion, while the levels of enzyme continued to increase, suggesting that the VDE enzyme does not turn over rapidly. The level of VDE changed in an inverse, nonlinear relationship with respect to the VAZ pool, suggesting that enzyme levels could be indirectly regulated by the VAZ pool.
Mechanically Induced Avoidance Response of Chloroplasts in Fern Protonemal Cells
Authors: Sato Yoshikatsu , Kadota Akeo , Wada Masamitsu 
Published: July 2002

Abstract
Cell response to mechanical stimulation was investigated at a subcellular level in protonemal cells of the fern Adiantum capillus-veneris L. by pressing a small part of the cell with a microcapillary. In cells receiving local stimulation, the chloroplasts moved away from the site of stimulation, whereas the nuclei failed to show such avoidance movement. Mechanical stimulation for a period as short as 0.3 min was enough to induce the avoidance response to a maximal level. The avoidance movement of chloroplasts started within 30 min and the plateau level of avoidance was attained around 2 h after stimulation. By tracing the movement of chloroplasts during the response, it was shown that the mobility of chloroplasts near the stimulation site increased transiently within 1 h after the stimulation. After 2 to 3 h, it slowed down to the control level without stimulation. The avoidance response was inhibited by 0.1 mm cytochalasin B and 25 mm2,3-butanedione monoxime but not by 3.3 μmamiprophosmethyl or 5 mm colchicine. These findings indicate that the protonemal cells were very sensitive to mechanical stimulation and that chloroplasts moved away from the mechanically stimulated site through the actomyosin motile system.
Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastoris
Authors: Mertens Jeffrey A. , Shiraishi Naomasa , Campbell Wilbur H. 
Published: July 2002

Abstract
Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO3 reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L−1, corn MoR at 32 mg L−1, and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L−1. Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.
The Mid-Pericarp Cell Layer in Soybean Pod Walls Is a Multicellular Compartment Enriched in Specific Lipoxygenase Isoforms
Authors: Dubbs Wesley E. , Grimes Howard D. 
Published: July 2002

Abstract
Specific lipoxygenase isoforms immunolocalize to the cytosol of a single cell layer in the soybean (Glycine max L.) pod wall. The cells of this layer, termed the mid-pericarp layer (MPL), are larger than adjacent cells and are highly branched. The entire MPL appears to form an elaborate interdigitated network within the pod wall. A particularly striking feature of the MPL is the presence of extensive regions of very thin, approximately 30 nm, cell wall, which connect the cells of the MPL. It was demonstrated that after mechanical wounding of the pod wall, 40-kD fluorescein-dextran was able to move throughout the MPL. In addition, when pod walls are cut, an exudate flows from the MPL that is highly enriched in lipoxygenase isoforms (approximately 40% of the total protein). The MPL of soybean pod walls may represent a novel multicellular compartment involved in defense of leguminous plants.
Aspartate Kinase 2. A Candidate Gene of a Quantitative Trait Locus Influencing Free Amino Acid Content in Maize Endosperm
Authors: Wang Xuelu , Stumpf David K. , Larkins Brian A. 
Published: July 2002

Abstract
The maize (Zea mays) Oh545o2inbred accumulates an exceptionally high level of free amino acids, especially lysine (Lys), threonine (Thr), methionine, and iso-leucine. In a cross between Oh545o2 and Oh51Ao2, we identified several quantitative trait loci linked with this phenotype. One of these is on the long arm of chromosome 2 and is linked with loci encoding aspartate (Asp) kinase 2 and Asp kinase (AK)-homoserine dehydrogenase (HSDH) 2. To investigate whether these enzymes can contribute to the high levels of Asp family amino acids, we measured their specific activity and feedback inhibition properties, as well as activities of several other key enzymes involved in Lys metabolism. We did not find a significant difference in total activity of dihydrodipicolinate synthase, HSDH, and Lys ketoglutarate reductase between these inbreds, and the feedback inhibition properties of HSDH and dihyrodipicolinate synthase by Lys and/or Thr were similar. The most significant difference we found between Oh545o2 and Oh51Ao2 is feedback inhibition of AK by Lys but not Thr. AK activity in Oh545o2 is less sensitive to Lys inhibition than that in Oh51Ao2, with a Lys I50 twice that of Oh51Ao2. AK activity in Oh545o2 endosperm is also higher than in Oh51Ao2 at 15 d after pollination, but not 20 d after pollination. The results indicate that the Lys-sensitive Asp kinase 2, rather than the Thr-sensitive AK-HSDH2, is the best candidate gene for the quantitative trait locus affecting free amino acid content in Oh545o2.
Phytochrome A Mediates Blue Light and UV-A-Dependent Chloroplast Gene Transcription in Green Leaves
Authors: Chun Louis , Kawakami Alana , Christopher David A. 
Published: July 2002

Abstract
We characterized the photobiology of light-activated chloroplast transcription and transcript abundance in mature primary leaves by using the following two systems: transplastomic promoter-reporter gene fusions in tobacco (Nicotiana tabacum), and phytochrome (phyA, phyB, and hy2) and cryptochrome (cry1) mutants of Arabidopsis. In both dicots, blue light and UV-A radiation were the major signals that activated total chloroplast and psbA, rbcL, and 16S rrntranscription. In contrast, transcription activities in plants exposed to red and far-red light were 30% to 85% less than in blue light/UV-A, depending on the gene and plant species. Total chloroplast,psbA, and 16S rrn transcription were 60% to 80% less in the Arabidopsis phyA mutant exposed to blue light/UV-A relative to wild type, thus definitively linking phyA signaling to these photoresponses. To our knowledge, the major role of phyA in mediating the blue light/UV-A photoresponses is a new function for phyA in chloroplast biogenesis at this stage of leaf development. AlthoughrbcL expression in plants exposed to UV-A was 50% less in the phyA mutant relative to wild type, blue light-induced rbcL expression was not significantly affected in the phyA, phyB, andcry1 mutants. However, rbcL expression in blue light was 60% less in the phytochrome chromophore mutant,hy2, relative to wild type, indicating that another phytochrome species (phyC, D, or E) was involved in blue light-inducedrbcL transcription. Therefore, at least two different phytochromes, as well as phytochrome-independent photosensory pathways, mediated blue light/UV-A-induced transcription of chloroplast genes in mature leaves.
Regulation of Alternative Oxidase Activity in Six Wild Monocotyledonous Species. An in Vivo Study at the Whole Root Level
Authors: Millenaar Frank F. , Gonzàlez-Meler Miquel A. , Fiorani Fabio , Welschen Rob , Ribas-Carbo Miquel , Siedow James N. , Wagner Anneke M. , Lambers Hans 
Published: July 2002

AbstractThe activity of the alternative pathway is affected by a number of factors, including the level and reduction state of the alternative oxidase (AOX) protein, and the reduction state of the ubiquinone pool. To investigate the significance of these factors for the rate of alternative respiration in vivo, we studied root respiration of six wild monocotyledonous grass species that were grown under identical controlled conditions. The activity of the alternative pathway was determined using the oxygen isotope fractionation technique. In all species, the AOX protein was invariably in its reduced (high activity) state. There was no correlation between AOX activity and AOX protein concentration, ubiquinone (total, reduced, or oxidized) concentration, or the reduction state of the ubiquinone pool. However, when some of these factors are combined in a linear regression model, a good fit to AOX activity is obtained. The function of the AOX is still not fully understood. It is interesting that we found a positive correlation between the activity of the alternative pathway and relative growth rate; a possible explanation for this correlation is discussed. Inhibition of the AOX (with salicylhydroxamic acid) decreases respiration rates less than the activity present before inhibition (i.e. measured with the 18O-fractionation technique).
shl, a New Set of Arabidopsis Mutants with Exaggerated Developmental Responses to Available Red, Far-Red, and Blue Light
Authors: Pepper Alan E. , Seong-Kim Mi-seon , Hebst Stephanie M. , Ivey Kathryn N. , Kwak Su-Jin , Broyles Denise E. 
Published: July 2002

AbstractThe interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light—a threshold condition in which photomorphogenetic signaling pathways are only partially active—for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light.shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant andphyB mutant backgrounds but may be dependent onCRY1 for phenotypic expression in blue light. Theshl2 phenotype was partially dependent onPHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent onHY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared byCRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC,PHYD, and PHYE).
Application of high‐precision isotope ratio monitoring mass spectrometry to identify the biosynthetic origins of proteins
Authors: Apostol Izydor , Brooks Paul D. , Mathews Antony J. 
Published: July 2002

AbstractIsotope ratio monitoring (IRM) mass spectrometry was used to measure the relative abundance of stable isotopes in several samples of adult human hemoglobin expressed in E. coli, yeast, and human blood. The results showed significant differences in the distribution of 15N and 13C isotopes among hemoglobin samples produced in these organisms. This indicates that IRM mass spectrometry can be used in forensic protein chemistry to identify the origin of protein expression.
An Improved Transfer Matrix Method for Steady-State Analysis of
Nonlinear Rotor-Bearing Systems
Authors: Zu J. W. , Ji Z. 
Published: July 2002

Abstract
An improved transfer matrix method is developed to analyze nonlinear rotor-bearing systems. The rotating shaft is described by the Timoshenko beam theory which considers the effect of the rotary inertia and shear deformation. A typical roller bearing model is assumed which has cubic nonlinear spring and linear damping characteristics. Transfer matrices for the Timoshenko shaft element, disk element, and nonlinear bearing element are derived and the global transfer matrix is formed. The steady-state response of synchronous, subharmonic, and superharmonic whirls is determined using the harmonic balance method. Two numerical examples are presented to demonstrate the effectiveness of this approach.
Persistence ofChlamydia trachomatisIs Induced by Ciprofloxacin and Ofloxacin In Vitro
Authors: Dreses-Werringloer Ute , Padubrin Ingrid , Jürgens-Saathoff Barbara , Hudson Alan P. , Zeidler H. , Köhler L. 
Published: July 2002

ABSTRACTAn in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection withChlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steady-state levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations forC. trachomatisallows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.
Failure of Short-Course Ceftriaxone Chemotherapy for Multidrug-Resistant Typhoid Fever in Children: a Randomized Controlled Trial in Pakistan
Authors: Bhutta Zulfiqar A. , Khan Iqtidar A. , Shadmani Mamoon 
Published: July 2002

ABSTRACT

The precise duration of therapy of multidrug-resistant (MDR) typhoid with broad-spectrum cephalosporins is uncertain. We prospectively randomized 57 children with culture-proven MDR typhoid to receive treatment with intravenous ceftriaxone (CRO) (65 mg/kg of body weight/day) for 7 days (short course;
n
= 29) or 14 days (conventional;
n
= 28). The response to therapy, as evaluated by the serial monitoring of the typhoid morbidity score and bacteriological clearance, was comparable between groups. In contrast to the conventional therapy, 14% of the children receiving CRO for 7 days had a confirmed bacteriological relapse within 4 weeks of stopping therapy.
In Vitro Activities of Methylenecyclopropane Analogues of Nucleosides and Their Phosphoralaninate Prodrugs against Cytomegalovirus and Other Herpesvirus Infections
Authors: Rybak Rachel J. , Hartline Caroll B. , Qiu Yao-Ling , Zemlicka Jiri , Harden Emma , Marshall Gwen , Sommadossi Jean-Pierre , Kern Earl R. 
Published: July 2002

ABSTRACT

Human cytomegalovirus (HCMV) infection does not generally cause problems in the immunocompetent adult but can result in severe clinical disease in the fetus, neonate, and immunocompromised host. Ganciclovir (GCV), the agent currently used to treat most HCMV infections, has resulted in much therapeutic success; however, efficacy remains suboptimal. Therefore, there is still a need to develop new compounds for use against HCMV infections. In the present study, several
Z
- and
E
-series methylenecyclopropane analogues and their phosphoroalaninate prodrugs were tested initially for activity against HCMV, strain AD169, and murine cytomegalovirus (MCMV) in vitro. Many were found to exhibit efficacy comparable to that of GCV against HCMV in plaque assays and were active against MCMV as well. The compounds were also tested for efficacy against herpes simplex virus types 1 and 2, varicella-zoster virus, and Epstein-Barr virus, and some had levels of activity that were comparable to that of acyclovir. In addition, the compounds synguanol (QYL-438) and 2-amino-6-cyclopropylamino analogue (QYL-769) were chosen for further evaluation and were found to be effective against additional laboratory and clinical isolates of HCMV and GCV-resistant isolates. QYL-438 and QYL-769 were found to be nontoxic in human and mouse fibroblasts and were considerably less toxic than GCV in granulocyte macrophage CFUs and erythroid burst-forming units. These results provide evidence for the high activity of some of these methylenecyclopropane analogues against various herpesviruses, particularly HCMV, in tissue culture and suggest that further evaluation is warranted to determine their potential for use in future clinical studies.