bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Profiling Reveals Novel Interactions between Wounding, Pathogen, Abiotic Stress, and Hormonal Responses in Arabidopsis,
Authors: Cheong Yong Hwa , Chang Hur-Song , Gupta Rajeev , Wang Xun , Zhu Tong , Luan Sheng 
Published: July 2002

Abstract
Mechanical wounding not only damages plant tissues, but also provides pathways for pathogen invasion. To understand plant responses to wounding at a genomic level, we have surveyed the transcriptional response of 8,200 genes in Arabidopsis plants. Approximately 8% of these genes were altered by wounding at steady-state mRNA levels. Studies of expression patterns of these genes provide new information on the interactions between wounding and other signals, including pathogen attack, abiotic stress factors, and plant hormones. For example, a number of wound-responsive genes encode proteins involved in pathogen response. These include signaling molecules for the pathogen resistance pathway and enzymes required for cell wall modification and secondary metabolism. Many osmotic stress- and heat shock-regulated genes were highly responsive to wounding. Although a number of genes involved in ethylene, jasmonic acid, and abscisic acid pathways were activated, many in auxin responses were suppressed by wounding. These results further dissected the nature of mechanical wounding as a stress signal and identified new genes that may play a role in wounding and other signal transduction pathways.
Novel Insight into Vascular, Stress, and Auxin-Dependent and -Independent Gene Expression Programs in Strawberry, a Non-Climacteric Fruit
Authors: Aharoni Asaph , Keizer Leopold C.P. , Van Den Broeck Hetty C. , Blanco-Portales Rosario , Muñoz-Blanco Juan , Bois Gregory , Smit Patrick , De Vos Ric C.H. , O'Connell Ann P. 
Published: July 2002

Abstract
Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria × ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.
Proteome Analysis of Grain Filling and Seed Maturation in Barley
Authors: Finnie Christine , Melchior Sabrina , Roepstorff Peter , Svensson Birte 
Published: July 2002

Abstract
In monocotyledonous plants, the process of seed development involves the deposition of reserves in the starchy endosperm and development of the embryo and aleurone layer. The final stages of seed development are accompanied by an increase in desiccation tolerance and drying out of the mature seed. We have used two-dimensional gel electrophoresis for a time-resolved study of the changes in proteins that occur during seed development in barley (Hordeum vulgare). About 1,000 low-salt extractable protein spots could be resolved on the two-dimensional gels. Protein spots were divided into six categories according to the timing of appearance or disappearance during the 5-week period of comparison. Nineteen different proteins or protein fragments in 36 selected spots were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MS) or nano-electrospray tandem MS/MS. Some proteins were present throughout development (for example, cytosolic malate dehydrogenase), whereas others were associated with the early grain filling (ascorbate peroxidase) or desiccation (Cor14b) stages. Most noticeably, the development process is characterized by an accumulation of low-M r α-amylase/trypsin inhibitors, serine protease inhibitors, and enzymes involved in protection against oxidative stress. We present examples of proteins not previously experimentally observed, differential extractability of thiol-bound proteins, and possible allele-specific spot variation. Our results both confirm and expand on knowledge gained from previous analyses of individual proteins involved in grain filling and maturation.
Two-Component Signal Transduction Pathways in Arabidopsis
Authors: Hwang Ildoo , Chen Huei-Chi , Sheen Jen 
Published: July 2002

Abstract
The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.
Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases
Authors: Zhou Jiming , Hartmann Stefanie , Shepherd Brianne K. , Poulton Jonathan E. 
Published: July 2002

Abstract
In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1,Ph2, Ph3, Ph4, andPh5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.
SPINDLY Is a Nuclear-Localized Repressor of Gibberellin Signal Transduction Expressed throughout the Plant
Authors: Swain Stephen M. , Tseng Tong-Seung , Thornton Tina M. , Gopalraj Manjula , Olszewski Neil E. 
Published: July 2002

Abstract
SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing aSPY::GUS reporter gene reveals thatSPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, andgai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A secondSPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue thespy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/orO-N-acetyl-glucosamine modification of these proteins.
Contrapuntal Networks of Gene Expression during Arabidopsis Seed Filling[W]
Authors: Ruuska Sari A. , Girke Thomas , Benning Christoph , Ohlrogge John B. 
Published: July 2002

Abstract
We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant wrinkled1 (wri1) seeds that have an 80% reduction in oil. Between 5 and 13 days after flowering, a period preceding and including the major accumulation of storage oils and proteins, ∼35% of the genes represented on the array changed at least twofold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to the biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 days after flowering. By contrast, the expression of storage proteins, oleosins, and other known abscisic acid–regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins, implicating a role in CO2 refixation and the supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic to plastid metabolism. Despite major changes in metabolism between wri1 and wild-type seeds, &lt;1% of genes differed by more than twofold, and most of these were involved in central lipid and carbohydrate metabolism. Thus, these data define in part the downstream responses to disruption of the WRI1 gene.
The ArabidopsisSKU5Gene Encodes an Extracellular Glycosyl Phosphatidylinositol–Anchored Glycoprotein Involved in Directional Root Growth[W]
Authors: Sedbrook John C. , Carroll Kathleen L. , Hung Kai F. , Masson Patrick H. , Somerville Chris R. 
Published: July 2002

AbstractTo investigate how roots respond to directional cues, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.
Crystal structures of free, IMP‐, and GMP‐bound Escherichia coli hypoxanthine phosphoribosyltransferase
Authors: Guddat Luke W. , Vos Siska , Martin Jennifer L. , Keough Dianne T. , de Jersey John 
Published: July 2002

AbstractCrystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5′‐monophosphate (IMP) and guanosine 5′‐monophosphate (GMP) (2.8 Å resolution). Of the known 6‐oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Cα atoms of 1.0 Å. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6‐oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6‐oxopurine PRTases have a requirement for divalent metal ion, usually Mg2+ in vivo. In the free structure, a Mg2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6‐oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2‐exocyclic nitrogen of guanine (in the HPRT‐guanine‐PRib‐PP‐Mg2+ complex). However, hypoxanthine does not have a 2‐exocyclic atom and the HPRT‐IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine‐binding pocket.
Efficient docking of peptides to proteins without prior knowledge of the binding site
Authors: Hetényi Csaba , van der Spoel David 
Published: July 2002

AbstractReliability in docking of ligand molecules to proteins or other targets is an important challenge for molecular modeling. Applications of the docking technique include not only prediction of the binding mode of novel drugs, but also other problems like the study of protein‐protein interactions. Here we present a study on the reliability of the results obtained with the popular AutoDock program. We have performed systematical studies to test the ability of AutoDock to reproduce eight different protein/ligand complexes for which the structure was known, without prior knowledge of the binding site. More specifically, we look at factors influencing the accuracy of the final structure, such as the number of torsional degrees of freedom in the ligand. We conclude that the Autodock program package is able to select the correct complexes based on the energy without prior knowledge of the binding site. We named this application blind docking, as the docking algorithm is not able to “see” the binding site but can still find it. The success of blind docking represents an important finding in the era of structural genomics.
Structural basis for cyclodextrins' suppression of human growth hormone aggregation
Authors: Otzen Daniel Erik , Knudsen Benjamin Raerup , Aachmann Finn , Larsen Kim Lambertsen , Wimmer Reinhard 
Published: July 2002

AbstractMany therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various β‐CD derivatives is sufficient to completely avoid this. α‐ and γ‐CD are considerably less effective. Stopped‐flow data on the aggregation reaction in the presence of β‐CD are analyzed according to a minimalist association model to yield an apparent hGH‐β‐CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence‐based titration of hGH with β‐CD. Nuclear magnetic resonance studies indicate that β‐CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side‐chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.
The neuropeptide Y monomer in solution is not folded in the pancreatic‐polypeptide fold
Authors: Bettio Andrea , Dinger Michaela C. , Beck‐Sickinger Annette G. 
Published: July 2002

AbstractFluorescence‐labelled analogs of NPY, a 36‐amino acid peptide amide, were synthesized by solid‐phase peptide synthesis and used for fluorescence‐resonance energy transfer studies to investigate the conformation. Energy‐transfer efficiency measurements in different media at the concentration of 10 μM are in agreement with a model of the NPY structure proposed by NMR studies (performed at millimolar concentration) in which the C‐terminal part of the molecule adopts an α‐helical conformation while the N‐terminal part is flexible. According to this model, the α‐helix is stabilized by intermolecular hydrophobic interactions because of the formation of dimers. The decrease of the peptide concentration causes a shift of the dimerization equilibrium toward the monomeric form. Energy‐transfer efficiency measurements performed at lower concentrations do not support the hypothesis of the folding back of the N‐terminal tail onto the C‐terminal α‐helix to yield the so‐called “PP‐fold” conformation. This structure is observed in the crystal structure of avian pancreatic polypeptide, a member of the NPY peptide hormone family, and it has been considered to be the bioactive one. Our results complete the structural characterization of NPY in solution at concentration ranges in which NMR experiments are not feasible. Furthermore, these results open the way to study the conformation of the receptor‐bound ligand.
Characterization of ostrich (Struthio camelus) β‐microseminoprotein (MSP): Ideication of homologous sequences in EST databases and analysis of their evolution during speciation
Authors: Lazure Claude , Villemure Michéle , Gauthier Dany , Naudé Ryno J. , Mbikay Majambu 
Published: July 2002

Abstractβ‐Microseminoprotein, alternatively called prostatic secretory protein of 94 amino acids, is a hydrophilic, unglycosylated, small protein rich in conserved half‐cystine residues. Originally found in human seminal plasma and prostatic fluids, its presence was later shown in numerous secretions and its homologs were described in many vertebrate species. These studies showed that this protein had rapidly evolved, but they failed to unambiguously idey its biological role. Here, we show that a protein isolated from ostrich pituitary gland is closely related to a similar one isolated from chicken serum and that the two are structurally related to the mammalian β‐microseminoprotein. The complete 90–amino acid sequence of the ostrich molecule was established through a combination of automated Edman degradation and matrix‐assisted laser desorption ionization–time of flight (MALDI‐TOF) mass spectrometric procedures, including postsource decay (PSD) and ladder sequencing analyses. This study documents for the first time that β‐microseminoprotein is present in aves. It is also the first report of a C‐terminal amidated form for a member of this protein family and the first in which the disulfide linkages are established. Database searches using the herein‐described amino acid sequence allowed ideication of related proteins in numerous species such as cow, African clawed frog, zebrafish, and Japanese flounder. These small proteins show a strikingly high rate of amino acid substitutions, especially across phyla boundaries. Noticeably, no β‐microseminoprotein–related gene could be found in the recently completed fruit fly genome, indicating that if such a gene exists in arthropods, it must have extensively diverged from the vertebrate ones.
Native‐state conformational dynamics of GART: A regulatory pH‐dependent coil–helix transition examined by electrostatic calculations
Authors: Morikis Dimitrios , Elcock Adrian H. , Jennings Patricia A. , McCammon J. Andrew 
Published: July 2002

AbstractGlycinamide ribonucleotide transformylase (GART) undergoes a pH‐dependent coil–helix transition with pKa ∼ 7. An α‐helix is formed at high pH spanning 8 residues of a 21‐residue‐long loop, comprising the segment Thr120–His121–Arg122–Gln123–Ala124–Leu125–Glu126–Asn127. To understand the electrostatic nature of this loop–helix, called the activation loop–helix, which leads to the formation and stability of the α‐helix, pKa values of all ionizable residues of GART have been calculated, using Poisson–Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pKa values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to idey pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop–helix, and the impact of the activation helix on the catalytic site. Based on our pKa calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil–helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix‐promoting sequence Arg122–Gln123–Ala124–Leu125–Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic‐site residue His108, to facilitate catalysis.