bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calcium-Mediated Signaling during Sandalwood Somatic Embryogenesis. Role for Exogenous Calcium as Second Messenger
Authors: Anil Veena S. , Rao K. Sankara 
Published: July 2002

Abstract
The possible involvement of Ca2+-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. 45Ca2+-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher45Ca2+ incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca2+]cyt of PEMs, increasing from a resting concentration of 30 to 50 nm to 650 to 800 nm. Chelation of exogenous Ca2+ with ethyleneglycol-bis(aminoethyl ether)-N,N′-tetraacetic acid arrests such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca2+-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment withN-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving sandalwood Ca2+-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca2+ during sandalwood somatic embryogenesis.
Ethylene Perception by the ERS1 Protein in Arabidopsis
Authors: Hall Anne E. , Findell Jennifer L. , Schaller G. Eric , Sisler Edward C. , Bleecker Anthony B. 
Published: July 2002

Abstract
Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1(ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.
Radial Distribution Pattern of Pectin Methylesterases across the Cambial Region of Hybrid Aspen at Activity and Dormancy
Authors: Micheli Fabienne , Sundberg Björn , Goldberg Renée , Richard Luc 
Published: July 2002

Abstract
Biochemical microanalysis combined with tangential cryosectioning was used to visualize the distribution of pectin methylesterases (PMEs) across the cambial region in active and dormant hybrid aspen (Populus tremula L. × Populus tremuloides Michx). These novel techniques allowed us to relate activity and isoforms of PMEs to specific tissues and developmental stages of the stem to get more information on the physiological function of PMEs in cambial growth. Isoelectrofocusing analysis revealed numerous isoforms that were differentially distributed according to the tissue-type and to the cambial stage. A neutral isoform was found to be distributed ubiquitously across the stem of both active and dormant trees, which suggests that it is a housekeeping isoform involved in the maintenance of the cell wall integrity throughout the stem. In addition, two distinct isoforms having different isoelectric points were found to be related to the differentiation of cambial derivatives. A basic isoform appears to be a physiological marker of the dormant stage involved in the cessation of meristematic radial growth, whereas an acidic isoform is functionally related to the immediate expansion of the cambial daughter cells that occurs bilaterally on each side of the cambium at the active stage.
The Arabidopsis UVH1 Gene Is a Homolog of the Yeast Repair Endonuclease RAD1
Authors: Fidantsef Ana Lena , Mitchell David Lewis , Britt Anne Bagg 
Published: July 2002

Abstract
Ultraviolet radiation induces DNA damage products, largely in the form of pyrimidine dimers, that are both toxic and mutagenic. In most organisms, including Arabidopsis, these lesions are repaired both through a dimer-specific photoreactivation mechanism and through a less efficient light-independent mechanism. Several mutants defective in this “dark repair” pathway have been previously described. The mechanism of this repair has not been elucidated, but is thought to be homologous to the nucleotide excision repair mechanisms found in other eukaryotes. Here we report the complementation of the Arabidopsisuvh1 dark repair mutant with the Arabidopsis homolog of the yeast nucleotide excision repair gene RAD1, which encodes one of the subunits of the 5′-repair endonuclease. Theuvh1-2 mutant allele carries a glycine→aspartate amino acid change that has been previously identified to produce a null allele of RAD1 in yeast. Although Arabidopsis homologs of genes involved in nucleotide excision repair are readily identified by searching the genomic database, it has not been established that these homologs are actually required for dark repair in plants. The complementation of the Arabidopsisuvh1 mutation with the Arabidopsis RAD1homolog clearly demonstrates that the mechanism of nucleotide excision repair is conserved among the plant, animal, and fungal kingdoms.
Localization of a Nod Factor-Binding Protein in Legume Roots and Factors Influencing Its Distribution and Expression
Authors: Kalsi Gurpreet , Etzler Marilynn E. 
Published: July 2002

Abstract
The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (Db-LNP) that binds to the Nod factor signals produced by rhizobia that nodulate this plant. In this study we show that Db-LNP is differentially distributed along the surface of the root axis in a pattern that correlates with the zone of nodulation of the root. Db-LNP is present on the surface of young and emerging root hairs and redistributes to the tips of the root hairs in response to treatment of the roots with a rhizobial symbiont or with a carbohydrate ligand. This redistribution does not occur in response to a non-symbiotic rhizobial strain or a root pathogen. Db-LNP is also present in the root pericycle where its level decreases upon initiation of nodule formation. Maximum levels of Db-LNP are found in 2-d-old roots, and the expression of this root protein is increased when the plants are grown in the absence of NO3 − and NH4 +. These results support the possibility that Db-LNP is involved in the initiation of theRhizobium legume symbiosis.
Dynamic Localization of Rop GTPases to the Tonoplast during Vacuole Development
Authors: Lin Yakang , Seals Darren F. , Randall Stephen K. , Yang Zhenbiao 
Published: July 2002

Abstract
Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.
A Role for Inositol 1,4,5-Trisphosphate in Gravitropic Signaling and the Retention of Cold-Perceived Gravistimulation of Oat Shoot Pulvini
Authors: Perera Imara Y. , Heilmann Ingo , Chang Soo Chul , Boss Wendy F. , Kaufman Peter B. 
Published: July 2002

Abstract
Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP3) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP3 levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP3 levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP3 were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP3, and reduced gravitropic bending by 65%. Short-term changes in InsP3 were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4°C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP3 were present at 4°C. We propose a role for InsP3 in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP3 may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.
Cellular and Subcellular Localization ofS-Adenosyl-l-Methionine:Benzoic Acid Carboxyl Methyltransferase, the Enzyme Responsible for Biosynthesis of the Volatile Ester Methylbenzoate in Snapdragon Flowers
Authors: Kolosova Natalia , Sherman Debra , Karlson Dale , Dudareva Natalia 
Published: July 2002

Abstract
The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed byS-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.
Eukaryotic Peptide Deformylases. Nuclear-Encoded and Chloroplast-Targeted Enzymes in Arabidopsis
Authors: Dirk Lynnette M.A. , Williams Mark A. , Houtz Robert L. 
Published: July 2002

Abstract
Arabidopsis (ecotype Columbia-0) genes,AtDEF1and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-lengthAtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed inEscherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.
Macromolecular organization of the Yersinia pestis capsular F1 antigen: Insights from time‐of‐flight mass spectrometry
Authors: Tito Mark A. , Miller Julie , Griffin Kate F. , Williamson E. Diane , Titball Richard W. , Robinson Carol V. 
Published: July 2002

AbstractMass spectrometry has been used to examine the subunit interactions in the capsular F1 antigen from Yersinia pestis, the causative agent of the plague. Introducing the sample using nanoflow electrospray from solution conditions in which the protein remains in its native state and applying collisional cooling to minimize the internal energy of the ions, multiple subunit interactions have been maintained. This methodology revealed assemblies of the F1 antigen that correspond in mass to both 7‐mers and 14‐mers, consistent with interaction of two seven‐membered units. The difference between the calculated masses and those measured experimentally for these higher‐order oligomers was found to increase proportionately with the size of the complex. This is consistent with a solvent‐filled central cavity maintained on association of the 7‐mer to the 14‐mer. The charge states of the ions show that an average of one and four surface accessible basic side‐chains are involved in maintaining the interactions between the 7‐mer units and neighboring subunits, respectively. Taken together, these findings provide new information about the stoichiometry and packing of the subunits involved in the assembly of the capsular antigen structure. More generally, the data show that the symmetry and packing of macromolecular complexes can be determined solely from mass spectrometry, without any prior knowledge of higher order structure
HMG‐D complexed to a bulge DNA: An NMR model
Authors: Cerdan Rachel , Payet Dominique , Yang Ji‐Chun , Travers Andrew A. , Neuhaus David 
Published: July 2002

AbstractAn NMR model is presented for the structure of HMG‐D, one of the Drosophila counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA2 DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high‐resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA2 bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non‐sequence‐specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG–DNA complex structures are discussed.
Heat capacity changes upon burial of polar and nonpolar groups in proteins
Authors: Loladze Vakhtang V. , Ermolenko Dmitri N. , Makhatadze George I. 
Published: July 2002

AbstractIn this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, ΔCp, that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the 1H‐15N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in ΔCp are several times larger than those predicted, based on the changes in water accessible surface area upon substitution.
CODA: A combined algorithm for predicting the structurally variable regions of protein models
Authors: Deane Charlotte M. , Blundell Tom L. 
Published: July 2002

AbstractCODA, an algorithm for predicting the variable regions in proteins, combines FREAD a knowledge based approach, and PETRA, which constructs the region ab initio. FREAD selects from a database of protein structure fragments with environmentally constrained substitution tables and other rule‐based filters. FREAD was parameterized and tested on over 3000 loops. The average root mean square deviation ranged from 0.78 Å for three residue loops to 3.5 Å for eight residue loops on a nonhomologous test set. CODA clusters the predictions from the two independent programs and makes a consensus prediction that must pass a set of rule‐based filters. CODA was parameterized and tested on two unrelated separate sets of structures that were nonhomologous to one another and those found in the FREAD database. The average root mean square deviation in the test set ranged from 0.76Å for three residue loops to 3.09 Å for eight residue loops. CODA shows a general improvement in loop prediction over PETRA and FREAD individually. The improvement is far more marked for lengths six and upward, probably as the predictive power of PETRA becomes more important.CODA was further tested on several model structures to determine its applicability to the modeling situation. A web server of CODA is available at http://www‐cryst.bioc.cam.ac.uk/∼charlotte/Coda/search_coda.html.
Amino‐acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability
Authors: Krowarsch Daniel , Otlewski Jacek 
Published: July 2002

AbstractIt is widely accepted that solvent‐exposed sites in proteins play only a neglible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD‐ and DSC‐monitored stability: The Tden difference between the least (P1 Trp) and the most stable (P1 His) mutant is 11.2°C at pH 2.0. The ΔHden versus Tden plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (ΔCp,den = 0.41 kcal• mole−1 • K−1; 1 cal = 4.18 J) leading to a ΔGden difference of 2.1 kcal•mole−1. Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (ΔTden =7.1°C). Selective reduction of the Cys14–Cys 38 disulfide bond, which is located in the vicinity of the P1 position did not eliminate the stability differences. A correlation analysis of the P1 stability with different properties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein‐stability‐derived secondary structure propensity scales should be taken with more caution.