bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Spinal Antinociceptive Effect of Nocistatin in Neuropathic Rats Is Blocked by D-Serine
Authors: Muth-Selbach Uta , Dybek Eva , Kollosche Katrin , Stegmann Jens-Ulrich , Holthusen Holger , Lipfert Peter , Zeilhofer Hanns Ulrich 
Published: August 2004

Background
The neuropeptide nocistatin (NST) has been implicated in the modulation of nociceptive responses in the spinal cord. Depending on the dose, both pronociceptive and antinociceptive effects have repeatedly been reported. The pronociceptive effect is most likely attributable to inhibition of synaptic glycine and gamma-aminobutyric acid release and a subsequent reduction in the activation of inhibitory glycine and gamma-aminobutyric acid receptors, but the mechanisms of its antinociceptive action have hitherto remained elusive. It has recently been demonstrated that synaptically released glycine contributes to N-methyl-D-aspartate receptor activation. The authors therefore investigated whether a reduction in glycine release might also account for the antinociceptive action of NST in neuropathic rats.

Methods
The authors analyzed the effects of spinally applied NST in the chronic constriction injury model of neuropathic pain. NST was injected intrathecally from nanomolar to picomolar doses and its effects on thermal paw withdrawal latencies were monitored. Furthermore, we tested whether D-serine (100 microg per rat), a full agonist at the glycine binding site of the N-methyl-D-aspartate receptor, would interfere with the effects of NST.

Results
At high doses (10 nmol/rat), intrathecally injected NST was pronociceptive, whereas lower doses (1 pmol/rat) elicited antinociception. The antinociceptive, but not the pronociceptive, action was occluded by intrathecal pretreatment with D-serine. L-serine, which does not bind to N-methyl-D-aspartate receptors, affected neither the pronociceptive nor the antinociceptive effect.

Conclusions
These results demonstrate that NST produces a biphasic dose-dependent effect on neuropathic pain. The spinal antinociception by NST is most likely attributable to inhibition of glycine-dependent N-methyl-D-aspartate receptor activation.
Organ Polarity in Arabidopsis. NOZZLE Physically Interacts with Members of the YABBY Family
Authors: Sieber Patrick , Petrascheck Michael , Barberis Alcide , Schneitz Kay 
Published: August 2004

Abstract
Plant lateral organs exhibit proximal-distal and adaxial-abaxial polarity. In Arabidopsis, abaxial cell fate is regulated in part by putative transcription factors of the YABBY family, such as FILAMENTOUS FLOWER (FIL) and INNER NO OUTER (INO), by a mechanism that currently is not fully understood. NOZZLE (NZZ) encodes a plant-specific nuclear protein. Genetic evidence has shown that NZZ is involved in the positive feedback regulation of INO, thereby acting both as a temporal and spatial repressor of INO transcription. This mechanism allows the ovule primordium to complete its proximal-distal organization, prior to the onset of adaxial-abaxial development in the chalaza. During our study, we isolated FIL in a yeast two-hybrid screen using NZZ as bait. In vitro pull-down experiments confirmed the NZZ-FIL interaction. NZZ also bound INO and YABBY3, suggesting that NZZ generally interacts with YABBY proteins in vitro. The polar-charged region of NZZ was necessary and sufficient to bind to the zinc finger of INO and to interact with its C terminus carrying the high mobility group-like domain. We suggest that NZZ coordinates proximal-distal patterning and adaxial-abaxial polarity establishment in the developing ovule by directly binding to INO.
Roles for Class III HD-Zip and KANADI Genes in Arabidopsis Root Development
Authors: Hawker Nathaniel P. , Bowman John L. 
Published: August 2004

Abstract
Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.
Graft Transmission of a Floral Stimulant Derived from CONSTANS
Authors: Ayre Brian G. , Turgeon Robert 
Published: August 2004

Abstract
Photoperiod in plants is perceived by leaves and in many species influences the transition to reproductive growth through long-distance signaling. CONSTANS (CO) is implicated as a mediator between photoperiod perception and the transition to flowering in Arabidopsis. To test the role of CO in long-distance signaling, CO was expressed from a promoter specific to the companion cells of the smallest veins of mature leaves. This expression in tissues at the inception of the phloem translocation stream was sufficient to accelerate flowering at the apical meristem under noninductive (short-day) conditions. Grafts that conjoined the vegetative stems of plants with different flower-timing phenotypes demonstrated that minor-vein expression of CO is able to substitute for photoperiod in generating a mobile flowering signal. Our results suggest that a CO-derived signal(s), or possibly CO itself, fits the definition of the hypothetical flowering stimulant, florigen.
Dynamic Changes in the Osmotic Water Permeability of Protoplast Plasma Membrane
Authors: Moshelion Menachem , Moran Nava , Chaumont François 
Published: August 2004

Abstract
The osmotic water permeability coefficient (Pf) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial Pf, Pf rate-of-change (slopePf), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The Pf during the delay was ≤1 μm s−1. During the swelling period following the delay, Pf changed dynamically: within the first 15 s Pf either (1) increased gradually to approximately 8 μm s−1 (in the majority population of low-initial-Pf cells) or (2) increased abruptly to 10 to 20 μm s−1 and then decreased gradually to 3 to 6 μm s−1 (in the minority population of high-initial-Pf cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial Pf values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the Pf in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing Pf increase, thereby confirming the hypothesis that Pf dynamics, delay included, reflected the varying activity of aquaporins.
Brassinosteroids Do Not Undergo Long-Distance Transport in Pea. Implications for the Regulation of Endogenous Brassinosteroid Levels
Authors: Symons Gregory M. , Reid James B. 
Published: August 2004

Abstract
It is widely accepted that brassinosteroids (BRs) are important regulators of plant growth and development. However, in comparison to the other classical plant hormones, such as auxin, relatively little is known about BR transport and its potential role in the regulation of endogenous BR levels in plants. Here, we show that end-pathway BRs in pea (Pisum sativum) occur in a wide range of plant tissues, with the greatest accumulation of these substances generally occurring in the young, actively growing tissues, such as the apical bud and young internodes. However, despite the widespread distribution of BRs throughout the plant, we found no evidence of long-distance transport of these substances between different plant tissues. For instance, we show that the maintenance of steady-state BR levels in the stem does not depend on their transport from the apical bud or mature leaves. Similarly, reciprocal grafting between the wild type and the BR-deficient lkb mutants demonstrates that the maintenance of steady-state BR levels in whole shoots and roots does not depend on either basipetal or acropetal transport of BRs between these tissues. Together, with results from 3H-BR feeding studies, these results demonstrate that BRs do not undergo long-distance transport in pea. The widespread distribution of end-pathway BRs and the absence of long-distance BR transport between different plant tissues provide significant insight into the mechanisms that regulate BR homeostasis in plants.
Rapid Regulation of the Methylerythritol 4-Phosphate Pathway during Isoprene Synthesis
Authors: Wolfertz Michael , Sharkey Thomas D. , Boland Wilhelm , Kühnemann Frank 
Published: August 2004

Abstract
More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d2) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d2 displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d2 completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K m of isoprene synthase. The results confirm that isoprene synthase has a K m 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.
Functional Characterization ofOsMADS18, a Member of theAP1/SQUASubfamily of MADS Box Genes
Authors: Fornara Fabio , Pařenicová Lucie , Falasca Giuseppina , Pelucchi Nilla , Masiero Simona , Ciannamea Stefano , Lopez-Dee Zenaida , Altamura Maria Maddalena , Colombo Lucia , Kater Martin M. 
Published: August 2004

AbstractMADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function.
The lys5 Mutations of Barley Reveal the Nature and Importance of Plastidial ADP-Glc Transporters for Starch Synthesis in Cereal Endosperm
Authors: Patron Nicola J. , Greber Boris , Fahy Brendan F. , Laurie David A. , Parker Mary L. , Denyer Kay 
Published: August 2004

Abstract
Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.
Relocalization of Nuclear ALY Proteins to the Cytoplasm by the Tomato Bushy Stunt Virus P19 Pathogenicity Protein
Authors: Uhrig Joachim F. , Canto Tomas , Marshall David , MacFarlane Stuart A. 
Published: August 2004

AbstractThe P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity.
Biological activity, membrane‐targeting modification, and crystallization of soluble human decay accelerating factor expressed inE. coli
Authors: White Jennifer , Lukacik Petra , Esser Dirk , Steward Michael , Giddings Naomi , Bright Jeremy R. , Fritchley Sarah J. , Morgan B. Paul , Lea Susan M. , Smith Geoffrey P. , Smith Richard A.G. 
Published: August 2004

AbstractDecay‐accelerating factor (DAF, CD55) is a glycophosphatidyl inositol‐anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression inEscherichia coli, isolation and membrane‐targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C‐terminal cysteine residue to permit site‐specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed inPichia pastoris. Modification with a membrane‐localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF‐derived therapeutic. Crystals of theE. coli‐derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X‐ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.
Sensitivity of molecular dynamics simulations to the choice of the X‐ray structure used to model an enzymatic reaction
Authors: Garcia‐Viloca Mireia , Poulsen Tina D. , Truhlar Donald G. , Gao Jiali 
Published: August 2004

AbstractA subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X‐ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl‐CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton‐abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis.
Buried hydrophobic side‐chains essential for the folding of the parallel β‐helix domains of the P22 tailspike
Authors: Betts Scott , Haase‐Pettingell Cameron , Cook Kristen , King Jonathan 
Published: August 2004

AbstractThe processive β‐strands and turns of a polypeptide parallel β‐helix represent one of the topologically simplest β‐sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel β‐helix followed by an interdigitated section of triple‐stranded β‐helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two β‐helix domains and extend into the core of the contiguous triple β‐prism domain. To test whether these side‐chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel β‐helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple‐stranded β‐helix and contiguous β‐prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel β‐helix, triple‐stranded β‐helix, and β‐prism domains in the tailspike protein.
A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge
Authors: Kaderbhai Mustak A. , Davey Hazel M. , Kaderbhai Naheed N. 
Published: August 2004

AbstractA directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With‐in “sequence spaces” ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane‐associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of −1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore‐dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.
A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants
Authors: Verheyden Gert , Matrai Janka , Volckaert Guido , Engelborghs Yves 
Published: August 2004

AbstractThe kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of α‐chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped‐flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild‐type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild‐type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild‐type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.