bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved estimation of controlled direct effects in the presence of unmeasured confounding of intermediate variables
Authors: Kaufman Sol , Kaufman Jay S. , MacLehose Richard F. , Greenland Sander , Poole Charles 
Published: March 2005

AbstractAdjusting for a causal intermediate is a common analytic strategy for estimating an average causal direct effect (ACDE). The ACDE is the component of the total exposure effect that is not relayed through the specified intermediate. Even if the total effect is unconfounded, the usual ACDE estimate may be biased when an unmeasured variable affects the intermediate and outcome variables. Using linear programming optimization to compute non‐parametric bounds, we develop new ACDE estimators for binary measured variables in this causal structure, and use root mean square confounding bias (RMSB) to compare their performance with the usual stratified estimator in simulated distributions of target populations comprised of the 64 possible potential response types as well as distributions of target populations restricted to subsets of 18 or 12 potential response types defined by monotonicity or no‐interactions assumptions of unit‐level causal effects. We also consider target population distributions conditioned on fixed outcome risk among the unexposed, or fixed true ACDE in one stratum of the intermediate. Results show that a midpoint estimator constructed from the optimization bounds has consistently lower RMSB than the usual stratified estimator both unconditionally and conditioned on any risk in the unexposed. When conditioning on true ACDE, this midpoint estimator performs more poorly only when conditioned on an extreme true ACDE in one stratum of the intermediate, yet outperforms the stratified estimator in the other stratum when interaction is permitted. An alternate ‘limit‐modified crude’ estimator can never perform less favourably than the stratified estimator, and often has lower RMSB. Copyright © 2005 John Wiley & Sons, Ltd.
Pattern of changes in species diversity, structure and dynamics of forest ecosystems along latitudinal gradients in East Asia
Authors: Takyu Masaaki , Kubota Yasuhiro , Aiba Shin‐ichiro , Seino Tatsuyuki , Nishimura Takashi 
Published: March 2005

AbstractWe examined effects of seasonality of climate and dominant life form (evergreen/deciduous, broad‐leaf/coniferous) together with energy condition on species diversity, forest structure, forest dynamics, and productivity of forest ecosystems by comparing the patterns of changes in these ecosystem attributes along altitudinal gradients in tropical regions without seasonality and along a latitudinal gradient from tropical to temperate regions in humid East Asia. We used warmth index (temperature sum during growing season, WI) as an index of energy condition common to both altitudinal and latitudinal gradients. There were apparent differences in patterns of changes in the ecosystem attributes in relation to WI among four forest formations that were classified according to dominant life form and climatic zone (tropical/temperate). Many of the ecosystem attributes—Fisher's alpha of species‐diversity indices, maximum tree height and stem density, productivity [increment rate of aboveground biomass (AGB)], and population and biomass turnover rates—changed sharply with WI in tropical and temperate evergreen broad‐leaved forests, but did not change linearly or changed only loosely with WI in temperate deciduous broad‐leaved and evergreen coniferous forests. Values of these ecosystem attributes in temperate deciduous broad‐leaved and evergreen coniferous forests were higher (stem density was lower) than those in tropical and temperate evergreen broad‐leaved forests under colder conditions (WI below 100°C). Present results indicate that seasonality of climate and resultant change in dominant life form work to buffer the effects of energy reduction on ecosystem attributes along latitudinal gradients.
Rapid formation of amyloid from α‐monomeric recombinant human PrP in vitro
Authors: Tahiri‐Alaoui Abdessamad , James William 
Published: March 2005

AbstractThe infectious agent of prion diseases is identified with PrPSc, a β‐rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrPC. To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low‐level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP90–231 in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrPC‐like state in vitro, that the lag time of the reaction can be further shortened by the presence of a “seed” of pre‐formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.
Extraordinarily stable disulfide‐linked homodimer of human growth hormone
Authors: Grigorian Alexei L. , Bustamante Juan J. , Hernandez Peter , Martinez Andrew O. , Haro Luis S. 
Published: March 2005

AbstractAlthough a 22‐kDa human growth hormone (hGH) is the predicted protein product of the hGH‐Ngene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS‐PAGE under reducing conditions we isolated an unusually stable mercaptoethanol‐resistant (MER) 45‐kDa hGH. A 5‐h incubation at 100°C in the presence of 2‐mercaptoethanol was required to convert approximately 90% of MER‐45‐kDa hGH into a 22‐kDa hGH. Other reductants were not as effective in splitting MER‐45‐kDa hGH. After fracturing MER‐45‐kDa hGH, the 22‐kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N‐terminal residues were obtained for MER‐45‐kDa hGH and its 22‐kDa hGH cleavage product. Structural identity of MER‐45‐kDa hGH and 22‐kDa hGH was demonstrated by MALDI‐TOF mass spectrometry of tryptic digests. MER‐45‐kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER‐45‐kDa hGH is not a single chain polypeptide but is instead a homodimer of 22‐kDa hGH monomers. The MER‐45‐kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER‐45‐kDa hGH's interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.
ATP specifically drives refolding of non‐native conformations of cytochrome c
Authors: Sinibaldi Federica , Mei Giampiero , Polticelli Fabio , Piro M. Cristina , Howes Barry D. , Smulevich Giulietta , Santucci Roberto , Ascoli Franca , Fiorucci Laura 
Published: March 2005

AbstractAn increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid‐bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low‐spin species having distinct heme‐iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non‐native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.
l‐Arginine increases the solubility of unfolded species of hen egg white lysozyme
Authors: Reddy K. Ravi Charan , Lilie Hauke , Rudolph Rainer , Lange Christian 
Published: March 2005

Abstractl‐Arginine (l‐Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by l‐Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for unfolded and intermediate states during the process of oxidative refolding. An increased equilibrium solubility of unfolded species and intermediates in the presence of l‐Arg seems to be its major mechanism of action.
Rotational orientation of monomers within a designed homo‐oligomer transmembrane helical bundle
Authors: Howard Kathleen P. , Liu Wei , Crocker Evan , Nanda Vikas , Lear James , Degrado William F. , Smith Steven O. 
Published: March 2005

AbstractA peptide designed to form a homo‐oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using 2H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane‐solubilized version of the water‐soluble coiled‐coil GCN4‐P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The 2H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by inter‐helical hydrogen bonding of asparagines at positions 7 and 14.
Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase
Authors: Nadeau Owen W. , Gogol Edward P. , Carlson Gerald M. 
Published: March 2005

AbstractPhosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3‐MDa hexadecameric complex, (αβγδ)4. PhK comprises two arched octameric (αβγδ)2 lobes that are oriented back‐to‐back with overall D2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three‐dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three‐dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 Å. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90° instead of 68°, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.
Ca2+‐induced structural changes in phosphorylase kinase detected by small‐angle X‐ray scattering
Authors: Priddy Timothy S. , Macdonald Brian A. , Heller William T. , Nadeau Owen W. , Trewhella Jill , Carlson Gerald M. 
Published: March 2005

AbstractPhosphorylase kinase (PhK), a 1.3‐MDa (αβγδ)4 hexadecameric complex, is a Ca2+‐dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (αβγδ)2 octameric lobes that are oriented back‐to‐back with overall D2 symmetry and joined by connecting bridges. From chemical cross‐linking and electron microscopy, it is known that the binding of Ca2+ by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca2+ in the PhK complex in solution, small‐angle X‐ray scattering was performed on nonactivated and Ca2+‐activated PhK. Although the overall dimensions of the complex were not affected by Ca2+, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca2+‐induced conformational change. Computer‐generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca2+‐activated conformers of PhK are consistent with Ca2+‐induced conformational changes in both the lobes and the interlobal bridges.
Self‐cleavage of fusion protein in vivo using TEV protease to yield native protein
Authors: Shih Yan‐Ping , Wu Hui‐Chung , Hu Su‐Ming , Wang Ting‐Fang , Wang Andrew H.‐J. 
Published: March 2005

AbstractOverproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self‐cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.
Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding
Authors: Vanbelle Christophe , Halgand Frédéric , Cedervall Tommy , Thulin Eva , Åkerfeldt Karin S. , Laprévote Olivier , Linse Sara 
Published: March 2005

AbstractCalbindin D28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post‐translational modifications of human calbindin. The protein was studied at 37°C or 50°C in the presence or absence of Ca2+. One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h−1 at 50°C for Ca2+‐free calbindin. Deamidation is slower for the Ca2+‐saturated protein. The deamidation process leads to two Asp iso‐forms, regular Asp and iso‐Asp. The form with regular Asp 203 binds four Ca2+ ions with high affinity and positive cooperativity, i.e., in a very similar manner to non‐deamidated protein. The form with β‐aspartic acid (or iso‐Asp 203) has reduced affinity for two or three sites leading to sequential Ca2+ binding, i.e., the Ca2+‐binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca2+ ions with high affinity in a parallel manner and with positive cooperativity.
Modulation of cooperativity in Mycobacterium tuberculosis NADPH‐ferredoxin reductase: Cation‐and pH‐induced alterations in native conformation and destabilization of the NADP+‐binding domain
Authors: Bhatt Anant Narayan , Shukla Nidhi , Aliverti Alessandro , Zanetti Giuliana , Bhakuni Vinod 
Published: March 2005

AbstractFprA, a Mycobacterium tuberculosis NADPH‐ferredoxin reductase, consists of two structural domains, a FAD‐binding and a NADP‐binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD‐binding domain and release of the protein‐bound flavin. The NADP‐binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 μM MgCl2 or CaCl2 or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apoenzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP‐binding domain showed that cations and pH 6.0 destabilized only the heat‐stable NADP‐binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP‐binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three‐dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.
Crystal structure of a novel polyisoprenoid‐binding protein from Thermus thermophilus HB8
Authors: Handa Noriko , Terada Takaho , Doi‐Katayama Yukiko , Hirota Hiroshi , Tame Jeremy R.H. , Park Sam‐Yong , Kuramitsu Seiki , Shirouzu Mikako , Yokoyama Shigeyuki 
Published: March 2005

AbstractThe isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side‐chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI‐like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight‐stranded, antiparallel β‐barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.
The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment
Authors: Duong‐Ly Krisna C. , Nanda Vikas , DeGrado William F. , Howard Kathleen P. 
Published: March 2005

AbstractThe M2 protein from influenza A virus is a 97‐amino‐acid protein with a single transmembrane helix that forms proton‐selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from ∼15° to 38°. An important issue for understanding the structure of M2TM is the role of peptide–lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date—each defined by a different set of criteria and in a different environment—might provide snapshots of the distinct conformational states sampled by the protein.
Misfolded forms of glyceraldehyde‐3‐phosphate dehydrogenase interact with GroEL and inhibit chaperonin‐assisted folding of the wild‐type enzyme
Authors: Polyakova Oxana V. , Roitel Olivier , Asryants Regina A. , Poliakov Alexei A. , Branlant Guy , Muronetz Vladimir I. 
Published: March 2005

AbstractWe studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH‐groups modified by 5,5′‐dithiobis(2‐nitrobenzoate); (2) O‐R‐type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O‐P‐type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O‐R‐type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants Kd of 0.4 and 0.9 μM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg‐ATP. Chemically modified GAPDH and the O‐R‐type mutant dimers inhibited GroEL‐assisted refolding of urea‐denatured wild‐type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O‐R‐dimers, the O‐P‐type mutant dimers neither bound nor affected GroEL‐assisted refolding of the wild‐type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.