bluuebunny/crossref_metadata_2025_split huggingface dataset:
Action Spectrum for Cryptochrome-Dependent Hypocotyl Growth Inhibition in Arabidopsis
Authors: Ahmad Margaret , Grancher Nicholas , Heil Mary , Black Robert C. , Giovani Baldissera , Galland Paul , Lardemer Danielle 
Published: July 2002

Abstract
Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type,cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390–480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.
The Serine-Rich N-Terminal Domain of Oat Phytochrome A Helps Regulate Light Responses and Subnuclear Localization of the Photoreceptor
Authors: Casal Jorge J. , Davis Seth J. , Kirchenbauer Daniel , Viczian Andras , Yanovsky Marcelo J. , Clough Richard C. , Kircher Stefan , Jordan-Beebe Emily T. , Schäfer Eberhard , Nagy Ferenc , Vierstra Richard D. 
Published: July 2002

Abstract
Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Δ6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Δ6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Δ6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Δ6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Δ6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.
Differential Messenger RNA Gradients in the Unicellular AlgaAcetabularia acetabulum. Role of the Cytoskeleton
Authors: Vogel Heiko , Grieninger Gerd E. , Zetsche Klaus H. 
Published: July 2002

Abstract
The unicellular green alga Acetabularia acetabulum has proven itself to be a superior model for studies of morphogenesis because of its large size and distinctive polar morphology. The giant cell forms an elongated tube (a stalk of up to 60 mm in length), which at its apical pole makes whorls of hairs, followed by one whorl of gametophores in the shape of a cap. At its basal pole, the cell extends into a rhizoid wherein the single nucleus is positioned. In this study, we have determined the level of specific messenger RNAs in the apical, middle, and basal regions using reverse transcriptase-PCR methodology. Four mRNA classes were distinguished: those that were uniformly distributed (small subunit of Rubisco, actin-1, ADP-glucose, centrin, and α- and β-tubulin), those that expressed apical/basal (calmodulin-4) or basal/apical gradients (calmodulin-2 and a Ran-G protein), and those with development-specific patterns of distribution (mitogen-activated protein kinase, actin-2, and UDP-glucose-epimerase). Restoration of the apical/basal calmodulin-4 mRNA gradient after amputation of the apical region of the cell requires the nucleus and was abolished by cytochalasin D. Accumulation of actin-1 mRNA in the vicinity of the wound set by the amputation needs, likewise, the presence of the nucleus and was also inhibited by cytochalasin. This suggests that actin microfilaments of the cytoskeleton are involved in directed transport and/or anchoring of these mRNAs.
Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene
Authors: Hanaoka Hideki , Noda Takeshi , Shirano Yumiko , Kato Tomohiko , Hayashi Hiroaki , Shibata Daisuke , Tabata Satoshi , Ohsumi Yoshinori 
Published: July 2002

Abstract
Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion withinAtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in theatapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1plants. Senescence-associated genes SEN1 andYSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.
Overexpression of Cytosolic Glutamine Synthetase. Relation to Nitrogen, Light, and Photorespiration
Authors: Oliveira Igor C. , Brears Timothy , Knight Thomas J. , Clark Alexandra , Coruzzi Gloria M. 
Published: July 2002

Abstract
In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO2 photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO2 compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.
Microarray Analysis of Diurnal and Circadian-Regulated Genes in Arabidopsis
Authors: Schaffer Robert , Landgraf Jeff , Accerbi Monica , Simon Vernadette , Larson Matt , Wisman Ellen 
Published: July 2002

Abstract
Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY, CCA1, and GI, suggesting that other key circadian clock genes might be found within these clusters.
Molecular, crystallographic and algorithmic factors in structure determination from powder diffraction data by simulated annealing
Authors: Shankland Kenneth , McBride Lorraine , David William I. F. , Shankland Norman , Steele Gerald 
Published: July 2002

The crystal structure of famotidine form B has been solved directly from powder diffraction data by the application of simulated annealing. The molecule crystallizes in the monoclinic space groupP21/cwith refined unit-cell dimensionsa = 17.6547 (4),b= 5.2932 (1),c= 18.2590 (3) Å and β = 123.558 (1)° atT= 130 K. The core of this work is a systematic investigation of the influence of algorithmic, crystallographic and molecular factors on the structure solution process. With an appropriate choice of annealing schedule, molecular description and diffraction data range, the overall number of successes in solving the crystal structure is close to 100%. Other factors, including crystallographic search space restrictions and parameter sampling method, have little effect on the structure solution process. The basic principles elucidated here have been factored into the design of theDASHstructure solution program.
Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents
Authors: Byerly Douglas W. , McElroy Craig A. , Foster Mark P. 
Published: July 2002

AbstractIdentifying potential ligand binding sites on a protein surface is an important first step for targeted structure‐based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded 15N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand‐induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent‐induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR‐based screens.
Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore
Authors: Miles George , Bayley Hagan , Cheley Stephen 
Published: July 2002

AbstractThe gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(ΔCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell‐free in vitro transcription and translation system, and were shown to assemble into SDS‐stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC50 value of 1.64 ng mL−1, which is over 15 times more potent than staphylococcal α‐hemolysin. HlyII(ΔCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(ΔCT) on red cell membranes showed that the C‐terminal extension is sensitive to digestion, while HlyII(ΔCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I+80/I−80 = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (−160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal α‐hemolysin.
High‐throughput screening of soluble recombinant proteins
Authors: Shih Yan‐Ping , Kung Wen‐Mei , Chen Jui‐Chuan , Yeh Chia‐Hui , Wang Andrew H.‐J. , Wang Ting‐Fang 
Published: July 2002

AbstractThe aims of high‐throughput (HTP) protein production systems are to obtain well‐expressed and highly soluble proteins, which are preferred candidates for use in structure–function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96‐well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well‐expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high‐speed centrifugation in a 96‐tube format and analyzed by multiwell denaturing SDS‐PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome‐wide analysis.
Structural features underlying selective inhibition of protein kinase CK2 by ATP site‐directed tetrabromo‐2‐benzotriazole
Authors: Battistutta Roberto , De Moliner Erika , Sarno Stefania , Zanotti Giuseppe , Pinna Lorenzo A. 
Published: July 2002

AbstractTwo novel crystal structures of Zea mays protein kinase CK2α catalytic subunit, one in complex with the specific inhibitor 4,5,6,7‐tetrabromobenzotriazole (TBB) and another in the apo‐form, were solved at 2.2 Å resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2α is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five‐membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2α enzyme analyzed so far, with the exception of the complex with emodin. In the N‐terminal lobe, the position of helix αC is particularly well preserved in all the structures examined; the Gly‐rich loop is displaced from the intermediate position it has in the apo‐form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.
Transmembrane signal transduction of the αIIbβ3 integrin
Authors: Gottschalk Kay E. , Adams Paul D. , Brunger Axel T. , Kessler Horst 
Published: July 2002

AbstractIntegrins are composed of noncovalently bound dimers of an α‐ and a β‐subunit. They play an important role in cell‐matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low‐affinity and a high‐affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix–helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the αIIbβ3 integrin, this structural motif represents the high‐affinity state. A second conformation of the TM domain of αIIbβ3 is identified as the low‐affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three‐state mechanism.
Factors contributing to decreased protein stability when aspartic acid residues are in β‐sheet regions
Authors: Pokkuluri P.R. , Gu M. , Cai X. , Raffen R. , Stevens F.J. , Schiffer M. 
Published: July 2002

AbstractAsp residues are significantly under represented in β‐sheet regions of proteins, especially in the middle of β‐strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a β‐domain. Two Gln residues of the immunoglobulin light‐chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a β‐strand, and that of Q89D, located in the middle of a β‐strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild‐type VL‐Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL‐Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild‐type VL‐Len domain. The structures of mutants VL‐Len Q38D and VL‐Len Q89D/M4L/Y27dD/T94H were determined by X‐ray diffraction at 1.6 Å resolution. We found no major perturbances in the structures of these Q→D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main‐chain dihedral angles and in side‐chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a β‐sheet of an immunoglobulin‐type domain can have very serious consequences. A somatic mutation of a β‐strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.
Description of the topographical changes associated to the different stages of the DsbA catalytic cycle
Authors: Vinci Floriana , Couprie Joël , Pucci Piero , Quéméneur Eric , Moutiez Mireille 
Published: July 2002

AbstractThis paper provides a description of the surface topography of DsbA, the bacterial disulfide‐bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA‐substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease‐accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site.