bluuebunny/crossref_metadata_2025_split huggingface dataset:
Primary Structure and Expression of a 24-kD Vacuolar Protein (VP24) Precursor in Anthocyanin-Producing Cells of Sweet Potato in Suspension Culture
Authors: Xu Wenxin , Shioiri Hidenari , Kojima Mineo , Nozue Masayuki 
Published: July 2002

Abstract
A 24-kD vacuolar protein (VP24) accumulates abundantly in intravacuolar pigmented globules in anthocyanin-containing sweet potato (Ipomoea batatas) cells in suspension culture. A cDNA clone encoding VP24 was isolated from a cDNA library constructed from light-irradiated suspension-cultured cells. Sequence analysis revealed that a 2.9-kbp VP24 cDNA encodes a protein of 893 amino acid residues with a molecular mass of 96.3 kD. According to the deduced amino acid sequence of VP24 cDNA, VP24 is probably synthesized as a large precursor protein with an N-terminal extension composed of a signal peptide and a propeptide, plus the polypeptide of the mature VP24 and its C-terminal propeptide, which contains the multiple transmembrane domains. A search in the ProDom database revealed the mature VP24 domain belongs to the zinc metalloprotease family. Northern analysis revealed that the single 2.9-kb VP24 mRNA increases rapidly after light irradiation, whereas VP24 mRNA was undetectable in the dark-cultured cells or in the presence of a high concentration of 2,4-dichlorophenoxyacetic acid. Light-induced VP24 gene expression closely correlated with the accumulation of anthocyanin in the vacuoles. These results suggested that proteins derived from the VP24 precursor protein may be involved in vacuolar transport and/or accumulation of anthocyanin synthesized in the cytosol.
Evidence That Intragenic Recombination Contributes to Allelic Diversity of the S-RNase Gene at the Self-Incompatibility (S) Locus in Petunia inflata
Authors: Wang Xi , Hughes Austin L. , Tsukamoto Tatsuya , Ando Toshio , Kao Teh-Hui 
Published: July 2002

Abstract
For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population ofPetunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5′ region ofS 19 allele was derived from an ancestor allele closely related to S 2, whereas its 3′ region was derived from an ancestor allele closely related to S 8. A similar evolutionary relationship was found among S 3,S 12, andS 15 alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of theS 1 allele identified in this work is completely identical to that of the previously identifiedS 1 allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity,S 6 andS 9 alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed.
Hydrophobic Interactions of the Structural Protein GRP1.8 in the Cell Wall of Protoxylem Elements
Authors: Ringli Christoph , Hauf Gunter , Keller Beat 
Published: July 2002

AbstractThe glycine-rich structural protein GRP1.8 of French bean (Phaseolus vulgaris) is specifically localized in the modified primary cell walls of protoxylem elements. Continuous deposition of GRP1.8 into the cell walls during elongation growth of the plant suggests that GRP1.8 is part of a repair mechanism to strengthen the protoxylem. In this work, a reporter-protein system was developed to study the interaction of GRP1.8 with the extracellular matrix. Fusion proteins of a highly soluble chitinase with different domains of GRP1.8 were expressed in the vascular tissue of tobacco (Nicotiana tabacum), and the chemical nature of the interaction of these fusion proteins in the cell wall compartment was analyzed. In contrast with chitinase that required only low-salt conditions for complete extraction, the different chitinase/GRP1.8 fusion proteins were completely extracted only by a nonionic or ionic detergent, indicating hydrophobic interactions of GRP1.8. The same interactions were found with the endogenous GRP1.8 in bean hypocotyls. In addition, in vitro experiments indicate that oxidative cross-linking of tyrosines might account for the insolubilization of GRP1.8 observed in later stages of protoxylem development. Our data suggest that GRP1.8 forms a hydrophobic protein-layer in the cell wall of protoxylem vessels.
Mutator Transposase Is Widespread in the Grasses
Authors: Lisch Damon R. , Freeling Michael , Langham Richard J. , Choy Ming Y. 
Published: July 2002

Abstract
Although the Mutator (Mu) system is well characterized in maize (Zea mays), very little is known about this highly mutagenic system of transposons in other grasses. Mutator is regulated by the MuDRclass of elements, which encodes two genes, one of which,mudrA, has similarity to a number of bacterial transposases. Experiments in our laboratory, as well as database searches, demonstrate that mudrA sequences are ubiquitous and diverse in the grasses. In several species it is clear that multiple paralogous elements can be present in a single genome. In some species such as wheat (Triticum aestivum) and rice (Oryza sativa), mudrA-similar sequences are represented in cDNA databases, suggesting the presence of activeMu transposon systems in these species. Further, in rice and in sorghum, mudrA-like genes are flanked by long terminal inverted repeats, as well as the short host sequence direct repeats diagnostic of insertion. Thus, there is ample evidence that systems related to Mu in maize are at least potentially active in a wide variety of grasses. However, the mudrBgene, though important for Mu activity in maize, is not necessarily a component of Mu elements in other grasses.
Chloroplast and Mitochondrial Proteases in Arabidopsis. A Proposed Nomenclature
Authors: Adam Zach , Adamska Iwona , Nakabayashi Kazumi , Ostersetzer Oren , Haussuhl Kirsten , Manuell Andrea , Zheng Bo , Vallon Olivier , Rodermel Steven R. , Shinozaki Kazuo , Clarke Adrian K. 
Published: July 2002

AbstractThe identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.
Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress
Authors: Youssefian Shohab , Nakamura Michimi , Orudgev Emin , Kondo Noriaki 
Published: July 2002

Abstract
O-Acetylserine(thiol) lyase (OASTL), a key enzyme of plant sulfur metabolism, catalyzes the formation of Cys from sulfide and O-acetylserine. The biosynthesis of Cys is regarded as the exclusive function of sulfur reduction in plants, and a key limiting step in the production of glutathione (GSH), a thiol implicated in various cellular functions, including sulfur transport, gene expression, scavenging of reactive oxygen species (ROS), and resistance to biotic and abiotic stresses. To examine whether an increased capacity for cysteine (Cys) biosynthesis alters cellular responses to such stresses, we studied the differential changes in thiol levels and ROS scavenging of transgenic tobacco (Nicotiana tabacum) plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, to SO2 and to the ROS generator, methyl viologen. Intracellular Cys and GSH contents were generally higher incys1 transgenics than in controls under normal growth conditions, but became especially elevated in transgenic plants after SO2 exposure. An examination of differences in the ROS scavenging system of the transgenic plants also demonstrated the specific accumulation of Cu/Zn superoxide dismutase transcripts, known to be induced by Cys or GSH, and elevated cellular superoxide dismutase activities. The transgenic plants accordingly showed dramatic reductions in the extent of both foliar and photooxidative damage in response to acute SO2, as well as reduced levels of chlorosis and membrane damage following methyl viologen treatment. Overall, our results imply that OASTL plays a pivotal role in the synthesis of Cys and GSH that are required for regulation of plant responses to oxidative stress.
Mitochondrial Adaptations to NaCl. Complex I Is Protected by Anti-Oxidants and Small Heat Shock Proteins, Whereas Complex II Is Protected by Proline and Betaine
Authors: Hamilton E. William , Heckathorn Scott A. 
Published: July 2002

AbstractHigh soil sodium (Na) is a common stress in natural and agricultural systems. Roots are usually the first tissues exposed to Na stress and Na stress-related impairment of mitochondrial function is likely to be particularly important in roots. However, neither the effects of NaCl on mitochondrial function, nor its protection by several potential adaptive mechanisms, have been well studied. This study investigated the effects of NaCl stress on maize (Zea mays) mitochondrial electron transport and its relative protection by osmoprotectants (proline, betaine, and sucrose), antioxidants (ascorbate, glutathione, and α-tocopherol), antioxidant enzymes (catalase and Cu/Zn-superoxide dismutase), and mitochondrial small heat shock proteins (sHsps). We demonstrate that Complex I electron transport is protected by antioxidants and sHsps, but not osmoprotectants, whereas Complex II is protected only by low concentrations of proline and betaine. These results indicate that NaCl stress damaged Complex I via oxidative stress and suggests that sHsps may protect Complex I as antioxidants, but NaCl damaged Complex II directly. This is the first study to demonstrate that NaCl stress differentially affects Complex I and II in plants and that protection of Complex I and II during NaCl stress is achieved by different mechanisms.
The Catalytic Site of the Pectin Biosynthetic Enzyme α-1,4-Galacturonosyltransferase Is Located in the Lumen of the Golgi
Authors: Sterling Jason D. , Quigley Heather F. , Orellana Ariel , Mohnen Debra 
Published: July 2002

Abstract
α-1,4-Galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochromec reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1→4-linked α-d-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.
Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus aureus
Authors: Deivanayagam Champion C. S. , Perkins Samuel , Danthuluri Sita , Owens Rick T. , Bice Todd , Nanavathy Tamanna , Foster Timothy J. , Höök Magnus , Narayana Sthanam V. L. 
Published: July 2002

Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.39 and c = 112.65 Å. A complete data set was recorded to 2.1 Å resolution and had a Vm
of 2.3 Å3 Da−1 with 46.5% solvent, suggesting one molecule per asymmetric unit. Co-crystals of ClfA with the 17 amino-acid C-terminal peptide of fibrinogen γ-chain diffracted to 2.1 Å resolution and had unit-cell parameters a = 39.11, b = 81.39 and c = 109.51 Å in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit-cell parameters a = 96.31, b = 96.31 and c = 84.13 Å and diffracted to 2.45 Å resolution. The estimated Vm
of 2.6 Å3 Da−1 with 53% solvent indicated one molecule in the asymmetric unit.
Crystallization and preliminary X-ray diffraction studies of a novel bacterial esterase
Authors: Bourne Philip C. , Isupov Michail N. , Littlechild Jennifer A. 
Published: July 2002

A novel bacterial esterase has been crystallized in two forms suitable for X-ray diffraction studies. Crystals have been obtained by vapour-phase diffusion at 290 K using ammonium sulfate as precipitant. The first crystals grew in space group C2 with unit-cell parameters a = 134.7, b = 55.8, c = 110.3 Å, β = 125.1°. A monoclinic data set has been collected to 2.0 Å resolution. Microseeding yielded a second crystal form which grew in space group P212121 with unit-cell parameters a = 57.1, b = 115.4, c = 130.4 Å. Native data from these crystals have been collected to 1.6 Å resolution. A molecular envelope has been determined using an uranyl acetate derivative for phase calculation.
Structure of buffalo lactoferrin at 2.5 Å resolution using crystals grown at 303 K shows different orientations of the N and C lobes
Authors: Karthikeyan Subramanian , Paramasivam Murugan , Yadav Savita , Srinivasan Alagiri , Singh Tej P. 
Published: July 2002

The structure of buffalo lactoferrin has been determined at 303 K. The crystals belong to orthorhombic space group P212121, with unit-cell parameters a = 77.5, b = 91.0, c = 131.5 Å and Z = 4. The structure has been refined to an R factor of 0.187. The overall structure of the protein is similar to its structure determined at 277 K in a different crystal form. However, the lobe orientations in the two structures differ by 9.0°, suggesting significant inter-lobe flexibility in this family of proteins. The inter-lobe interactions are predominantly hydrophobic and could act as a cushion for a change in orientation under the influence of external conditions. On the other hand, the domain arrangements are found to be similar in 277 and 303 K crystal structures, with orientations differing by 1.5 and 1.0° in the N and C lobes, respectively. The results of these investigations suggest that the increase in temperature helps in the production of better quality crystals.
The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: Multiple forced covariant amino acid substitutions in natural variants
Authors: Deu Edgar , Koch Keith A. , Kirsch Jack F. 
Published: July 2002

AbstractThe role of the Lys68*Residues pertaining to the opposite subunit.
:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double‐mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of kcat/KM, L‐Asp and kcat/KM, α‐KG to salt bridge mutagenic replacements is shown by a linear‐free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, kcat/KM, L‐Asp and kcat/KM, α‐KG are 133 and 442 mM−1s−1 for the wild‐type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM−1s−1 for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.
Reduced antibody response to streptavidin through site‐directed mutagenesis
Authors: Meyer Damon L. , Schultz Jody , Lin Yukang , Henry Andrew , Sanderson James , Jackson Jeffrey M. , Goshorn Steve , Rees Anthony R. , Graves Scott S. 
Published: July 2002

AbstractStreptavidin provides an effective receptor for biotinylated tumoricidal molecules, including radionuclides, when conjugated to an antitumor antibody and administered systemically. Ideally, one would like to administer this bacterial protein to patients repeatedly, so as to maximize the antitumor effect without eliciting an immune response. Therefore, we attempted to reduce the antigenicity of streptavidin by mutating surface residues capable of forming high energy ionic or hydrophobic interactions. A crystallographic image of streptavidin was examined to identify residues with solvent‐exposed side chains and residues critical to streptavidin's structure or function, and to define loops. Mutations were incorporated cumulatively into the protein sequence. Mutants were screened for tetramer formation, biotin dissociation, and reduced immunoreactivity with pooled patient sera. Patient antisera recognized one minor continuous epitope with binding locus at residue E101 and one major discontinuous epitope involving amino acid residues E51 and Y83. Mutation of residues E51, Y83, R53, and E116 reduced reactivity with patient sera to <10% that of streptavidin, but these mutations were no less antigenic in rabbits. Mutant 37, with 10 amino acid substitutions, was only 20% as antigenic as streptavidin. Rabbits immunized with either streptavidin or mutant 37 failed to recognize the alternative antigen. Biotin dissociated from mutant 37 four to five times faster than from streptavidin. Residues were identified with previously undescribed impact on biotin binding and protein folding. Thus, substitution of charged, aromatic, or large hydrophobic residues on the surface of streptavidin with smaller neutral residues reduced the molecule's ability to elicit an immune response in rabbits.
Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin
Authors: Apiyo David , Wittung‐Stafshede Pernilla 
Published: July 2002

AbstractFlavodoxin is an α/β protein with a noncovalently bound flavin‐mononucleotide (FMN) cofactor. The apo‐protein adopts a structure identical to that of the holo‐form, although there is more dynamics in the FMN‐binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo‐flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo‐ and holo‐flavodoxins involve rather stable intermediates. In contrast, we here show that both holo‐ and apo‐forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two‐state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo‐protein. The folding and unfolding kinetics for holo‐flavodoxin exhibit two‐state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo‐flavodoxin, ∼280 μsec, is in excellent agreement with that predicted from the protein's native‐state topology. Unlike the holo‐protein behavior, the folding and unfolding reactions for apo‐flavodoxin are best described by two kinetic phases, with rates differing ∼15‐fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo‐flavodoxin are orders of magnitude slower (40‐ and 530‐fold, respectively) than that for the holo‐protein. We conclude that polypeptide–cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic‐folding path towards two‐state and speed up the folding reaction.
Pressure versus temperature unfolding of ribonuclease A: An FTIR spectroscopic characterization of 10 variants at the carboxy‐terminal site
Authors: Torrent Joan , Rubens Peter , Ribó Marc , Heremans Karel , Vilanova Maria 
Published: July 2002

AbstractFTIR spectroscopy was used to characterize and compare the temperature‐ and pressure‐induced unfolding of ribonuclease A and a set of its variants engineered in a hydrophobic region of the C‐terminal part of the molecule postulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two‐state, reversible unfolding transition using both pressure and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I′ band and the tyrosine band, or the maximum of the band assigned to the β‐sheet structure, corroborate the results of a previous study using fourth‐derivative UV absorbance spectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure‐ and temperature‐denaturated states of all the ribonucleases characterized retained some secondary structure. However, their spectral maxima were centered at different wavenumbers, which suggests that pressure‐ and temperature‐denaturated states do not have the same structural characteristics. Nevertheless, there was close correlation between the pressure and temperature midpoint transition values for the whole series of protein variants, which indicated a common tendency of stability toward pressure and heat.