bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Gas Exchange Measurement of the Site and Dynamics of Nitrate Reduction in Soybean
Authors: Cen Yan-Ping , Layzell David B. 
Published: March 2003

AbstractA gas analysis system was built to study the relationship between the reductant cost of NO3 − assimilation and the measured rate of CO2 and O2 exchange in roots, leaves, and stems+ petioles of soybean (Glycine max L. Merr. cv Maple glen) plants. The measurements were used to calculate the diverted reductant utilization rate (DRUR = 4*[measured rate of CO2 + measured rate of O2], in moles of high-energy electron [e −] per gram per hour) in plants in the presence (N+) and absence (N−) of NO3 −. The differences in DRUR between the N+ and N− treatments provided a measure of the NO3 −-coupled DRUR of 25-d-old plants, whereas a 15NO3 −-enriched nutrient solution was used to obtain an independent measure of the rate of NO3 − assimilation. The measured reductant cost for the whole plant was 9.6 e − per N assimilated, a value within the theoretical range of four to 10e − per N assimilated. The results predicted that shoots accounted for about 55% of the whole-plant NO3 − assimilation over the entire day, with shoots dominating in the light, and roots in the dark. The gas analysis approach described here holds promise as a powerful, noninvasive tool to study the regulation of NO3 − assimilation in plant tissue.
Noncovalent scFv multimers of tumor‐targeting anti‐Lewisy hu3S193 humanized antibody
Authors: Power Barbara E. , Doughty Larissa , Shapira Deborah R. , Burns John E. , Bayly Ann M. , Caine Joanne M. , Liu Zhanqi , Scott Andrew M. , Hudson Peter J. , Kortt Alexander A. 
Published: March 2003

AbstractSingle‐chain variable fragments (scFvs) of anti‐Lewisy hu3S193 humanized antibody were constructed by joining the VH and VL domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C‐terminal residues of the VH domain were removed (−1 residue, −2 residue) and then joined directly to the VL domain. An scFv construct in the reverse orientation with the VL joined directly to the VH domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non‐covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non‐covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.
Using nondenaturing mass spectrometry to detect fortuitous ligands in orphan nuclear receptors
Authors: Potier Noelle , Billas Isabelle M.L. , Steinmetz Anke , Schaeffer Christine , van Dorsselaer Alain , Moras Dino , Renaud Jean‐Paul 
Published: March 2003

AbstractNondenaturing electrospray mass spectrometry (ESI‐MS) has been used to reveal the presence of potential ligands in the ligand‐binding domain (LBD) of orphan nuclear receptors. This new approach, based on supramolecular mass spectrometry, allowed the detection and identification of fortuitous ligands for the retinoic acid‐related orphan receptor β (RORβ) and the ultraspiracle protein (USP). These fortuitous ligands were specifically captured from the host cell with the proper stoichiometry. After organic extraction, these molecules have been characterized by classic analytical methods and identified as stearic acid for RORβ and a phosphatidylethanolamine (PE) for USP, as confirmed by crystallography. These molecules act as “fillers” and may not be the physiological ligands, but they prove to be essential to stabilize the active conformation of the LBD, enabling its crystallization. The resulting crystal structures provide a detailed picture of the ligand‐binding pocket, allowing the design of highly specific synthetic ligands that can be used to characterize the function of orphan nuclear receptors. An additional advantage of this new method is that it is not based on a functional test and that it can detect low‐affinity ligands.
Denaturation and reassembly of chaperonin GroEL studied by solution X‐ray scattering
Authors: Arai Munehito , Inobe Tomonao , Maki Kosuke , Ikura Teikichi , Kihara Hiroshi , Amemiya Yoshiyuki , Kuwajima Kunihiro 
Published: March 2003

AbstractWe measured the denaturation and reassembly ofEscherichia colichaperonin GroEL using small‐angle solution X‐ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea‐induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil‐like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X‐ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.
Stable octameric structure of recombinant hemoglobin α2β283 Gly→Cys
Authors: Fablet Christophe , Marden Michael C. , Green Brian N. , Ho Chien , Pagnier Josée , Baudin‐Creuza Véronique 
Published: March 2003

AbstractWe have engineered a recombinant hemoglobin (rHb βG83C) based on the variant Hb Ta‐Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb βG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide‐binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto‐oxidation rate, and gel filtration experiments on the oligomeric βG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric βG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer βG83C was not modified after incubation with fresh plasma.
Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA
Authors: Matsumiya Shigeki , Ishino Sonoko , Ishino Yoshizumi , Morikawa Kosuke 
Published: March 2003

AbstractTwo mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site‐specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant PfuPCNAs were determined. Although the wild‐type PCNA forms a toroidal trimer with intermolecular hydrogen bonds between the N‐ and C‐terminal domains, the mutant PfuPCNAs exist as V‐shaped dimers through intermolecular hydrogen bonds between the two C‐terminal domains in the crystal. Because the mutated residues are involved in the intermolecular ion pairs through their side chains in the wild‐type PfuPCNA, these ion pairs seem to play a key role in maintaining the toroidal structure of the PfuPCNA trimer. The comparison of the crystal structures revealed intriguing conformational flexibility of each domain in the PfuPCNA subunit. This structural versatility of PCNA may be involved in the mechanisms for ring opening and closing.
How do helix–helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo‐oligomeric helical bundles
Authors: DeGrado William F. , Gratkowski Holly , Lear James D. 
Published: March 2003

AbstractThe final, structure‐determining step in the folding of membrane proteins involves the coalescence of preformed transmembrane helices to form the native tertiary structure. Here, we review recent studies on small peptide and protein systems that are providing quantitative data on the interactions that drive this process. Gel electrophoresis, analytical ultracentrifugation, and fluorescence resonance energy transfer (FRET) are useful methods for examining the assembly of homo‐oligomeric transmembrane helical proteins. These methods have been used to study the assembly of the M2 proton channel from influenza A virus, glycophorin, phospholamban, and several designed membrane proteins—all of which have a single transmembrane helix that is sufficient for association into a transmembrane helical bundle. These systems are being studied to determine the relative thermodynamic contributions of van der Waals interactions, conformational entropy, and polar interactions in the stabilization of membrane proteins. Although the database of thermodynamic information is not yet large, a few generalities are beginning to emerge concerning the energetic differences between membrane and water‐soluble proteins: the packing of apolar side chains in the interior of helical membrane proteins plays a smaller, but nevertheless significant, role in stabilizing their structure. Polar, hydrogen‐bonded interactions occur less frequently, but, nevertheless, they often provide a strong driving force for folding helix–helix pairs in membrane proteins. These studies are laying the groundwork for the design of sequence motifs that dictate the association of membrane helices.
A peptide model of insulin folding intermediate with one disulfide
Authors: Yan Han , Guo Zhan‐Yun , Gong Xiao‐Wen , Xi Dan , Feng You‐Min 
Published: March 2003

AbstractInsulin folds into a unique three‐dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single‐chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20–B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20‐B19]PIP) containing the single disulfide A20–B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2–3 magnitudes compared with that of the wild‐type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20–B19, which suggested that formation of the disulfide A20–B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild‐type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20–B19.
Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability
Authors: Sandgren Mats , Gualfetti Peter J. , Shaw Andrew , Gross Laurie S. , Saldajeno Mae , Day Anthony G. , Jones T. Alwyn , Mitchinson Colin 
Published: March 2003

AbstractAs part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (Tm) ranged from a 2.5°C increase to a 4.0°C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a Tm 7.7°C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o‐NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25°C to 60°C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild‐type T. reesei Cel12A enzyme. All of the structures have similar Cα traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.
Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization
Authors: Duval‐Terrié Caroline , Cosette Pascal , Molle Gérard , Muller Guy , Dé Emmanuelle 
Published: March 2003

AbstractThe aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C10) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain‐bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C10). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C10‐solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (α or β), 18C10 did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies.
Structural and kinetic characterization of the simplified SH3 domain FP1
Authors: Yi Qian , Rajagopal Ponni , Klevit Rachel E. , Baker David 
Published: March 2003

AbstractThe simplified SH3 domain sequence, FP1, obtained in phage display selection experiments has an amino acid composition that is 95% Ile, Lys, Glu, Ala, Gly. Here we use NMR to investigate the tertiary structure of FP1. We find that the overall topology of FP1 resembles that of the src SH3 domain, the hydrogen‐deuterium exchange and chemical shift perturbation profiles are similar to those of naturally occurring SH3 domains, and the 15N relaxation rates are in the range of naturally occurring small proteins. Guided by the structure, we further simplify the FP1 sequence and compare the effects on folding kinetics of point mutations in FP1 and the wild‐type src SH3 domain. The results suggest that the folding transition state of FP1 is similar to but somewhat less polarized than that of the wild‐type src SH3 domain.
A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in
Trichoplusia ni
Authors: Bull James C. , Godfray H. C. J. , O'Reilly David R. 
Published: April 2003

ABSTRACT

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral
fp25
gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of
Autographa californica
multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the
fp25
gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar
Trichoplusia ni
larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.
Identification, Timing, and Signal Specificity ofPseudomonas aeruginosaQuorum-Controlled Genes: a Transcriptome Analysis
Authors: Schuster Martin , Lostroh C. Phoebe , Ogi Tomoo , Greenberg E. P. 
Published: March 2003

ABSTRACTThere are two interrelated acyl-homoserine lactone quorum-sensing-signaling systems inPseudomonas aeruginosa. These systems, the LasR-LasI system and the RhlR-RhlI system, are global regulators of gene expression. We performed a transcriptome analysis to identify quorum-sensing-controlled genes and to better understand quorum-sensing control ofP. aeruginosagene expression. We compared gene expression in a LasI-RhlI signal mutant grown with added signals to gene expression without added signals, and we compared a LasR-RhlR signal receptor mutant to its parent. In all, we identified 315 quorum-induced and 38 quorum-repressed genes, representing about 6% of theP. aeruginosagenome. The quorum-repressed genes were activated in the stationary phase in quorum-sensing mutants but were not activated in the parent strain. The analysis of quorum-induced genes suggests that the signal specificities are on a continuum and that the timing of gene expression is on a continuum (some genes are induced early in growth, most genes are induced at the transition from the logarithmic phase to the stationary phase, and some genes are induced during the stationary phase). In general, timing was not related to signal concentration. We suggest that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression. Acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes duringP. aeruginosagrowth in culture.
Microarray Analysis of
Pseudomonas aeruginosa
Quorum-Sensing Regulons: Effects of Growth Phase and Environment
Authors: Wagner Victoria E. , Bushnell Daniel , Passador Luciano , Brooks Andrew I. , Iglewski Barbara H. 
Published: March 2003

ABSTRACT

Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the
las
and
rhl
systems, have been identified previously in the opportunistic pathogen
Pseudomonas aeruginosa
. High-density oligonucleotide microarrays for the
P. aeruginosa
PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying
las
and/or
rhl
QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [
N
-(3-oxododecanoyl) homoserine lactone and
N
-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (
P
≤ 0.05) in response to the exogenous autoinducers and were classified as QS regulated. A total of 244 genes were identified as being QS regulated at the mid-logarithmic phase, and 450 genes were identified as being QS regulated at the early stationary phase. Most of the previously reported QS-promoted genes were confirmed, and a large number of additional QS-promoted genes were identified. Importantly, 222 genes were identified as being QS repressed. Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated.
OmcB, ac-Type Polyheme Cytochrome, Involved in Fe(III) Reduction inGeobacter sulfurreducens
Authors: Leang Ching , Coppi M. V. , Lovley D. R. 
Published: March 2003

ABSTRACTMicroorganisms in the familyGeobacteraceaeare the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. TheGeobacter sulfurreducensgenome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designatedomcBandomcC, encode putative outer membrane polyhemec-type cytochromes which are 79% identical. The role of theomcBandomcCgenes in Fe(III) reduction inG. sulfurreducenswas investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. DisruptingomcBoromcCby gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. WhenomcBwas reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.