bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of an electroluminescent polymer and its non‐doped light‐emitting diodes with stable green emission
Authors: Liu Yingliang , Cao Huayu , Li Jianghui , Chen Zhijian , Xiao Lixin , Gong Qihuang , Cao Shaokui
Published: September 2008

An electroluminescent polymer was synthesized by Wittig condensation and characterized by the measurements of 1H‐NMR, IR, gel permeation chromatography (GPC), UV–Vis, PL, and cyclic voltammetry (CV). The polymer can be dissolved in common organic solvents such as tetrahydrofuran (THF), chloroform, and dichloromethane. The electroluminescent investigation showed that the non‐doped devices with a double‐layer configuration (ITO/PEDOT:PSS/Polymer/Mg:Ag) have a stable green emission property. The maximum luminance of the annealed device reaches 2317 cd/m2. The emission maximum and the CIE 1931 coordinate values are respectively stabilized at 552 nm and near (x, y) = (0.43, 0.55) with different voltages. Copyright © 2008 John Wiley & Sons, Ltd.
Biomedical applications of imidazolium cation‐modified iron oxide nanoparticles
Authors: Naka K. , Narita A. , Tanaka H. , Chujo Y. , Morita M. , Inubushi T. , Nishimura I. , Hiruta J. , Shibayama H. , Koga M. , Ishibashi S. , Seki J. , Kizaka‐Kondoh S. , Hiraoka M.
Published: September 2008

We report the synthesis of N‐methylimidazolium chloride‐modified iron oxide nanoparticles (MImCl‐NPs) by condensation of an imidazolium cation‐containing silane coupling agent (1) with the iron oxide nanoparticles which were prepared by the coprecipitation of FeCl2 and FeCl3 by alkaline hydrolysis. The modified iron oxide nanoparticles exhibited a superparamagnetic behavior at the room temperature. For a given weight concentration, MImCl‐NPs showed a darker (T2‐weighted) image than Resovist®, which is commercially available and used for MRI imaging in practice. When mice were given a daily intravenous injection of 5% glucose aqueous solution of MImCl‐NPs for two weeks, MImCl‐NPs showed no significant difference in body weight and blood biochemistry of serum in all samples compared to controls. DLS measurements of the aqueous solutions of the modified iron oxide nanoparticles after the addition of NaPF6 and NaI indicated the presence of particle aggregates. In contrast, no aggregation was observed after the addition of NaCl. A higher degree of adsorption of DNA onto MImCl‐NPs was observed in comparison to that onto amino‐functionalized magnetic nanoparticles (NH2‐NPs). Copyright © 2008 John Wiley & Sons, Ltd.
Short Twaron aramid fiber reinforced thermoplastic polyurethane
Authors: Akbarian M. , Hassanzadeh S. , Moghri M.
Published: September 2008

Mechanical, dynamic mechanical, and rheological behaviors of a short p‐aramid fiber reinforced thermoplastic polyurethane (TPU) have been studied in the range of 0–30 wt% of fibers. The tensile strength of the composite is improved slightly at higher fiber content with a minimum at around 10 wt% of fibers. The addition of fibers markedly reduces elongation at break and entails a steady increase in the elastic modulus, but decreases the wear resistance of the matrix. Storage modulus (E′) is increased and the shapes of loss tangent (tan δ) peaks point to a possible fiber–matrix interaction. Rheological studies show a power law behavior for all composites and increased viscosity with fiber loading. Study of the tensile and cryogenic fracture surfaces by scanning electron microscopy (SEM) indicates good correlation between the modes of failure and strength of the composites. The micrographs reveal good interfacial adhesion and extensive peeling and fibrillation of the fibers in the compounded and fractured composites. Theoretical models have been used to fit the experimental modulus data. Copyright © 2008 John Wiley & Sons, Ltd.
Cancer‐associated pH‐responsive tetracopolymeric micelles composed of poly(ethylene glycol)‐b‐poly(L‐histidine)‐b‐poly(L‐lactic acid)‐b‐poly(ethylene glycol)
Authors: Oh Kyung Taek , Lee Eun Seong
Published: September 2008

To create a novel vector for specifically delivering anticancer therapy to solid tumors, we used diafiltration to synthesize pH‐sensitive polymeric micelles. The micelles, formed from a tetrablock copolymer [poly(ethylene glycol)‐b‐poly(L‐histidine)‐b‐poly(L‐lactic acid)‐b‐poly(ethylene glycol)] consisted of a hydrophobic poly(L‐histidine) (polyHis) and poly(L‐lactic acid) (PLA) core and a hydrophilic poly(ethylene glycol) (PEG) shell, in which we encapsulated the model anticancer drug doxorubicin (DOX). The robust micelles exhibited a critical micellar concentration (CMC) of 2.1–3.5 µg/ml and an average size of 65–80 nm pH 7.4. Importantly, they showed a pH‐dependent micellar destabilization, due to the concurrent ionization of the polyHis and the rigidity of the PLA in the micellar core. In particular, the molecular weight of PLA block affected the ionization of the micellar core. Depending on the molecular weight of the PLA block, the micelles triggering released DOX at pH 6.8 (i.e. cancer acidic pH) or pH 6.4 (i.e. endosomal pH), making this system a useful tool for specifically treating solid cancers or delivering cytoplasmic cargoin vivo. Copyright © 2008 John Wiley & Sons, Ltd.
Microencapsulation of ammonium polyphosphate with PVA–melamine–formaldehyde resin and its flame retardance in polypropylene
Authors: Wu Kun , Song Lei , Wang Zhengzhou , Hu Yuan
Published: September 2008

With a shell of PVA–melamine–formaldehyde resin, microencapsulated ammonium polyphosphate (VMFAPP) is prepared by in situ polymerization and characterized by FTIR and XPS. Microencapsulation gives VMFAPP better water resistance and flame retardance compared with APP in PP. Thermal stability and fire resistance behavior have been analyzed and compared. The LOI value of the PP/VMFAPP composite is higher than that of the PP/APP composite. The UL 94 ratings of most of the PP/VMFAPP composites are V‐0, whereas PP/APP gives no rating at the 30% additive level. The water resistant properties of the PP composites are studied. Results of the cone calorimeter experiment show that VMFAPP is an effective flame retardant in PP compared with APP. The thermal degradation behaviors of APP and VMFAPP have been studied using TG and dynamic FTIR. Copyright © 2008 John Wiley & Sons, Ltd.
Exposure of the elderly to potential nephrotoxic drug combinations in Belgium
Authors: Smets Hilde LE , De Haes Johan FF , De Swaef André , Jorens Philippe G , Verpooten Gert A
Published: September 2008

Purpose

To count in the Belgian ambulatory prescription database Pharmanet, the number of elderly people treated with ACE inhibitors (ACEI) and/or angiotensine receptor blockers (ARB) (±diuretics ± β‐blockers) who eventually also received a prescription of non‐steroidal anti‐inflammatory drugs (NSAID) or spironolactone.

Method

All prescriptions dispensed in community pharmacies are collected in the Pharmanet database. The data of 2005 were used. Two age groups were studied: 65–79 years (label 1) and 80+ years (label 2). The following chronic treatments (subgroups) were studied: (A) ACEI/ARB; (B) ACEI/ARB + diuretics; (C) ACEI/ARB + spironolactone; (D) ACEI/ARB + β‐blocker + diuretic and (E) ACEI/ARB + β‐blocker + spironolactone.

Results

In 2005, 7.3% of Belgian population was chronically treated with ACEI/ARB. Twenty‐five per cent of 65–79 year‐old‐patients (subgroup A1) and 36.15% of 80+ year‐old‐patients (subgroup A2) received ACEI/ARB. At least one package of NSAID or spironolactone was prescribed to subgroup A1 in 25.44 and 5.80%, respectively, and to subgroup A2 in 22.04 and 9.89%, respectively. In all studied subgroups and age categories, NSAID were coprecribed in more than 20–35% of cases.

Conclusions

NSAID are frequently prescribed in elderly patients treated with ACEI or ARB in combination with diuretics. Severe renal adverse effects may result in this high‐risk population. Copyright © 2008 John Wiley & Sons, Ltd.
Potential population‐based electronic data sources for rapid pandemic influenza vaccine adverse event detection: a survey of health plans
Authors: Moore Kristen M. , Duddy April , Braun M. Miles , Platt Richard , Brown Jeffrey S.
Published: September 2008

Purpose

A vaccine against pandemic influenza may be rapidly and widely distributed, and could be used in populations with little prior exposure to influenza vaccines. Under such conditions, it will be important to gain timely information about the rates of vaccine adverse events, ideally by using electronic data from large populations. Many public and private health plans and payers have such information.

Methods

Between May and September 2007, we conducted a decision maker interview and technical assessment with several health plans in the United States. The interview and survey evaluated technical capability, organizational capacity, and willingness to participate in a coordinated program of rapid safety research targeting pandemic and other influenza vaccines.

Results

Eleven health plans (eight private, three public) participated in the decision maker interview. Most interviewees were medical directors or held similar positions within their organizations. Participating plans provided coverage and/or care for approximately 150 million members in the U.S. Nine health plans completed a technical assessment survey. Most decision makers indicated interest and willingness to participate in a coordinated rapid safety surveillance program, and all reported the necessary claims data analysis experience. Respondents noted legal, procedural, budgetary, and technical barriers to participation.

Conclusions

Senior decision makers representing private and public health plans were willing and asserted the ability of their organizations to participate in pandemic influenza vaccine safety monitoring. Developing working relationships, negotiating contracts, and obtaining necessary regulatory and legal approvals were identified as key barriers. These findings may be generalizable to other vaccines and pharmaceutical products. Copyright © 2008 John Wiley & Sons, Ltd.
Preparation and characterization of crosslinked resins containing ferrite particles
Authors: de Santa Maria Luiz Claudio , Simplício Simone , Ribeiro Carlos A.B. , Costa Marcos A.S. , Silva Manoel R. , Wang Shu H. , Amico Sandro C.
Published: September 2008

Micrometer‐sized magnetic particles hosted on network material were successfully prepared by a simple chemical process (ion exchange followed by co‐precipitation) from commercial styrene‐divinylbenzene copolymers. Energy dispersive X‐ray spectroscopy (EDS) coupled to scanning electron microscopy (SEM) allowed the observation of submicron particles. All the produced spherical beads have presented metallic particles (NiFe2O4, CuFe2O4, CoFe2O4, or MnFe2O4), either as isolated particles or agglomerates, located on their external and internal (within pores) The thermal stability of the composites, evaluated by thermogravimetric techniques, were found to be dependent on the amount of ferrite particles incorporated into them. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers.
Effect of surfactant type on the synthesis of PMMA using redox initiation and high frequency ultrasound
Authors: Parra Cristina , Albano Carmen , González Gema
Published: September 2008

In the present work, a study of the effect of the surfactant nature on the synthesis of polymethylmethacrylate (PMMA) obtained by high frequency ultrasonic radiation and redox initiation is presented. The surfactants used were an anionic: sodium lauryl sulfate (SLS) and a nonionic: nonylphenol etoxilated. Different morphologies, particle size, and microstructures were obtained depending on the surfactant concentration (0.5, 1.0, 1.5, and 2.0)%. The highest conversion values of PMMA were obtained with SLS, resulting in a highly syndiotactic polymer. High frequency ultrasound is very efficient when ionic surfactants are used. However, for nonionic surfactants ultrasonic irradiation is very inefficient to initiate polymerization, in this case the effect of redox initiation was very marked increasing polymerization conversion. For this, surfactant blends of PMMA/PEO were obtained when redox initiation was used together with high frequency ultrasound. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers.
Adhesion parameters at the interface in nanoparticulate filled polymer systems
Authors: Kovačević Vera , Vrsaljko Domagoj , Lučić Blagojević Sanja , Leskovac Mirela
Published: September 2008

The interface in particulate filled polymer systems determine to a large extent, the mechanical and morphological properties of composites and polymer blends. The present contribution describes surface free energy measurements and calculations of adhesion parameters at the interface in poly(vinyl acetate) (PVAc) and polyurethane (PU) composites as well as in styrene acrylonitrile/ethylene propylene diene monomer (SAN/EPDM) blends filled with calcium carbonate (CaCO3) nanofillers. The effects that a surface pretreatment of the filler with either silane or stearic acid had on the interface been investigated and related to the resulting properties of the composites and filled polymer blend. The optimal adhesion in the particulate composites and blends was determined by the minimal interfacial energy and optimal coefficient of wetting. The conditions of the optimal adhesion with filler location at the interface, predicting by the thermodynamical models, correlated well with the improvement of the morphological and mechanical properties. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers.
Synthesis of biodegradable polyhydroxyalcanoate copolymer from a renewable source by alternate feeding
Authors: Pereira Silvia M.F. , Sánchez Rubén J. , Rieumont Jacques , Cabrera Jose G.
Published: September 2008

Polyhydroxyalcanoates copolymers with 3‐hydroxybutirate (3HB) and 3‐hydroxyvalerate (3HV) co‐monomers, P3(HB‐co‐x%HV), were produced in fed‐batch culture by Ralstonia eutropha DSM428 using fructose as a single carbon source in the first step and adding propionic acid in the second step by alternating feeding. Polymer yield was 0.18 g/L with a content of 24 mol% of the 3HV fraction determined by 1H NMR. NMR measurements indicated that the polymer obtained is isotactic. The copolymer attained 35% of crystallinity according to X‐ray diffraction measurements, and two (020) planes were observed. Thermal behavior presented melting temperature at 154°C and the crystallization temperature was 65°C. A glass transition temperature was observed at −10°C. Average molecular weight measured by GPC was 4.9 × 105 Dalton. Isothermal radial growth rates of spherulites of P3(HB‐co‐24%HV) were studied. All experimental facts and the analysis of the sequence distribution of diads and triads of 3HB and 3HV units led to the conclusion that it is not a completely statistical random copolymer once it contains different types of segments. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers.
Photocrosslinkable hyperbranched polyfluorenes containing oxadiazole: synthesis, photophysics and electroluminescence
Authors: Tang Duo‐Feng , Wen Gui‐An , Wei Wei , Huang Wei
Published: September 2008

BACKGROUND: Crosslinkable hyperbranched polymers have great potential for use in organic light‐emitting devices and photovoltaics. With this in mind, two crosslinkable hyperbranched polyfluorenes, P5 and P6, containing oxadiazole as the core, and linear polyfluorene P0 were synthesized via palladium‐catalyzed Suzuki coupling. A device was also made.

RESULTS: The resulting polymers were characterized using NMR, gel permeation chromatography, differential scanning calorimetry and thermogravimetric analysis. The spectral behavior of the polymers was also investigated. Compared with uncrosslinked P5 and P6, the photoluminescence spectra of crosslinked P5 and P6 were little influenced by the crosslinking. After annealing for 3 h at 200 °C in air, the photoluminescence of P6 (with a greater content of branched units) showed excellent stability compared with that of P5 and P0. The results for the device revealed that photocrosslinking improves the stability of the electroluminescence.

CONCLUSION: The crosslinkable polymers obtained can be used in multilayer devices. Copyright © 2008 Society of Chemical Industry
Proteomics integrated with Escherichia coli vector‐based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase‐like protein of Propionibacterium acnes
Authors: Huang Cheng‐Po , Liu Yu‐Tsueng , Nakatsuji Teruaki , Shi Yang , Gallo Richard R. , Lin Shwu‐Bin , Huang Chun‐Ming
Published: September 2008

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time‐consuming task to examine a protein's immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet‐c) was used as a representative antigen to establish this platform. A cell wall‐anchoring sialidase‐like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector‐based vaccine by overexpressing Tet‐c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet‐c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector‐based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet‐c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV‐irradiated E. coli vector‐based vaccines. The antibody production of Tet‐c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes‐associated diseases.
Therapeutic starvation and autophagy in prostate cancer: A new paradigm for targeting metabolism in cancer therapy
Authors: DiPaola Robert S. , Dvorzhinski Dmitri , Thalasila Anu , Garikapaty Venkata , Doram Donyell , May Michael , Bray K. , Mathew Robin , Beaudoin Brian , Karp C. , Stein Mark , Foran David J. , White Eileen
Published: September 2008

BACKGROUND

Autophagy is a starvation induced cellular process of self‐digestion that allows cells to degrade cytoplasmic contents. The understanding of autophagy, as either a mechanism of resistance to therapies that induce metabolic stress, or as a means to cell death, is rapidly expanding and supportive of a new paradigm of therapeutic starvation.

METHODS

To determine the effect of therapeutic starvation in prostate cancer, we studied the effect of the prototypical inhibitor of metabolism, 2‐deoxy‐D‐glucose (2DG), in multiple cellular models including a transfected pEGFP‐LC3 autophagy reporter construct in PC‐3 and LNCaP cells.

RESULTS

We found that 2DG induced cytotoxicity in PC‐3 and LNCaP cells in a dose dependent fashion. We also found that 2DG modulated checkpoint proteins cdk4, and cdk6. Using the transfected pEGFP‐LC3 autophagy reporter construct, we found that 2DG induced LC3 membrane translocation, characteristic of autophagy. Furthermore, knockdown of beclin1, an essential regulator of autophagy, abrogated 2DG induced autophagy. Using Western analysis for LC3 protein, we also found increased LC3‐II expression in 2DG treated cells, again consistent with autophagy. In an effort to develop markers that may be predictive of autophagy, for assessment in clinical trials, we stained human prostate tumors for Beclin1 by immunohistochemistry (IHC). Additionally, we used a digitized imaging algorithm to quantify Beclin1 staining assessment.

CONCLUSIONS

These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials. Prostate © 2008 Wiley‐Liss, Inc.
Association of HPC2/ELAC2 and RNASEL non‐synonymous variants with prostate cancer risk in African American familial and sporadic cases
Authors: Robbins Christiane M. , Hernandez Wenndy , Ahaghotu Chiledum , Bennett James , Hoke Gerald , Mason Terry , Pettaway Curtis A. , Vijayakumar Srinivasan , Weinrich Sally , Furbert‐Harris Paulette , Dunston Georgia , Powell Isaac J. , Carpten John D. , Kittles Rick A.
Published: September 2008

Introduction

The RNASEL and HPC2/ELAC2 genes have been implicated in hereditary prostate cancer. Further assessment of the role of these genes in sporadic prostate cancer in African American men (AAM) is warranted.

Methods

Genotyping of HPC2/ELAC2 variants (S217L, A541T), along with RNASEL variants (R462Q and E541D) was completed in 155 African American sporadic and 88 familial prostate cancer cases, and 296 healthy male controls. Logistic regression analysis was performed and odds ratios (OR) were calculated, while correcting for both age and population stratification using admixture informative markers.

Results

The HPC2/ELAC2 217L allele was significantly associated with risk of prostate cancer when taking all cases into account (OR = 1.6; 1.0–2.6; P = 0.03). The RNASEL 541D allele was associated with a decrease in risk of prostate cancer in sporadic cases (OR = 0.4; 0.2–0.8; P = 0.01). We did not detect an association between prostate cancer risk and the RNASEL R462Q variant. Results from haplotype analyses of the two RNASEL variants revealed highly significant differences in haplotype allele frequencies between cases and controls suggesting a synergistic effect at the RNASEL locus. One haplotype in particular (462R–541D) is far more frequent in our control population and shows a strong protective effect against prostate cancer (OR = 0.47, P = 8.1 × 10−9).

Conclusions

These results suggest that HPC2/ELAC2 and RNASEL may play a role, however minor, in prostate cancer risk among AAM.
Insights into the DNA cleavage mechanism of human LINE‐1 retrotransposon endonuclease
Authors: Repanas Kostas , Fuentes Gloria , Cohen Serge X. , Bonvin Alexandre M. J. J. , Perrakis Anastassis
Published: September 2008

The human LINE‐1 endonuclease (L1‐EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1‐EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1‐EN residues—Asp145, Arg155, Ile204—that are key for DNA cleavage, and determined their crystal structures. The L1‐EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1‐EN, we performed molecular dynamics simulations using as model the structures of wild type EN‐L1, of two βB6‐βB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the βB6‐βB5 loop shows an anticorrelated motion with the surface loops βA6‐βA5 and βB3‐αB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped‐out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Towards accurate residue–residue hydrophobic contact prediction for α helical proteins via integer linear optimization
Authors: Rajgaria R. , McAllister S. R. , Floudas C. A.
Published: September 2008

A new optimization‐based method is presented to predict the hydrophobic residue contacts in α‐helical proteins. The proposed approach uses a high resolution distance dependent force field to calculate the interaction energy between different residues of a protein. The formulation predicts the hydrophobic contacts by minimizing the sum of these contact energies. These residue contacts are highly useful in narrowing down the conformational space searched by protein structure prediction algorithms. The proposed algorithm also offers the algorithmic advantage of producing a rank ordered list of the best contact sets. This model was tested on four independent α‐helical protein test sets and was found to perform very well. The average accuracy of the predictions (separated by at least six residues) obtained using the presented method was ∼66% for single domain proteins. The average true positive and false positive distances were also calculated for each protein test set and they are 8.87 and 14.67 Å, respectively. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Molecular dynamics of amicyanin reveals a conserved dynamical core for blue copper proteins
Authors: Rizzuti Bruno , Sportelli Luigi , Guzzi Rita
Published: September 2008

Molecular dynamics simulation has been carried out for the blue copper protein amicyanin from two different sources, Paracoccus denitrificans and Paraccocus versutus, to investigate the structural and dynamical properties common to the two molecules and to identify prominent features shared with proteins of the same family, the monomeric cupredoxins. The two amicyanins have almost identical secondary and tertiary structure. In the simulation, they differ for the number of hydrogen bonds in the main chain and the conformation of some β‐strands. However, they strictly maintain the arrangement of the portions of the β‐barrel that are conserved in the folding architecture of the blue copper proteins. Paracoccus versutus amicyanin equilibrates more rapidly, shows lower atomic deviation values, and is less rigid with respect to Paracoccus denitrificans amicyanin. Principal component analysis reveals that the conformational subspaces corresponding to eigenvectors with the same index for each of the two molecules are not necessarily equivalent. Nevertheless, a core scaffold with constrained dynamics exist for both amicyanins. In addition, two fairly flexible regions that are located on the opposite side with respect to the interaction sites with the partner molecules in the redox process have been evidenced in the protein structure. This description of amicyanin, with a few mobile regions remote from the active site and a rigid scaffold including most of the protein β‐barrel, has a close similarity with that of azurin and plastocyanin, two other cupredoxins previously investigated in simulation. Furthermore, similarities in the distribution of the atomic fluctuations indicate that amicyanin, azurin, and plastocyanin possess common dynamical features, in spite of differences in their structure. On the basis of these findings, we suggest that topological constraints imposed by the folding in correspondence of protein regions that are the most conserved determine the protein dynamics of the cupredoxin family. The dynamical properties of the cupredoxins might be controlled for functional advantages that include the binding mechanism with the biological partners and the collective inner motions of the protein matrix required for the electron transfer, whereas long‐range conformational changes in the redox reaction should be excluded. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Substrate directs enzyme dynamics by bridging distal sites: UDP‐galactopyranose mutase
Authors: Yao Xiaohui , Bleile Dustin W. , Yuan Yue , Chao Jay , Sarathy Karunan P. , Sanders David A. R. , Pinto B. Mario , O'Neill Melanie A.
Published: September 2008

UDP‐Galactopyranose mutase (UGM) is a flavoenzyme that catalyzes interconversion of UDP‐galactopyranose (UDP‐Galp) and UDP‐galactofuranose (UDP‐Galf); its activity depends on FAD redox state. The enzyme is vital to many pathogens, not native to mammals, and is an important drug target. We have probed binding of substrate, UDP‐Galp, and UDP to wild type and W160A UGM from K. pneumoniae, and propose that substrate directs recognition loop dynamics by bridging distal FAD and W160 sites; W160 interacts with uracil of the substrate and is functionally essential. Enhanced Trp fluorescence upon substrate binding to UGM indicates conformational changes remote from the binding site because the fluorescence is unchanged upon binding to W70F/W290F UGM where W160 is the sole Trp. MD simulations map these changes to recognition loop closure to coordinate substrate. This requires galactose‐FAD interactions as Trp fluorescence is unchanged upon substrate binding to oxidized UGM, or binding of UDP to either form of the enzyme, and MD show heightened recognition loop mobility in complexes with UDP. Consistent with substrate‐directed loop closure, UDP binds 10‐fold more tightly to oxidized UGM, yet substrate binds tighter to reduced UGM. This requires the W160‐U interaction because redox‐switched binding affinity of substrate reverses in the W160A mutant where it only binds when oxidized. Without the anchoring W160‐U interaction, an alternative binding mode for UDP is detected, and STD‐NMR experiments show simultaneous binding of UDP‐Galp and UDP to different subsites in oxidized W160A UGM: Substrate no longer directs recognition loop dynamics to coordinate tight binding to the reduced enzyme. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Fuzzy domains: New way of describing flexibility and interdependence of the protein domains
Authors: Yesylevskyy Semen O. , Kharkyanen Valery N.
Published: September 2008

We proposed the innovative method of domain identification based on the concept of the fuzzy domains. In this method each residue of the protein can belong to several domains simultaneously with certain weights, which reflect to what extent this residue shares the motion pattern of the given domain. Our method allows describing the fuzzy boundaries between the domains and the gradual changes of the motion pattern from one domain to the other. It provides the reasonable compromise between the continuous change of the protein dynamics from one residue to the other and the discrete description of the structure in terms of small number of domains. We suggested quantitative criterion, which shows the overall degree of domain flexibility in the protein. The concept of the fuzzy domains provides an innovative way of visualization of domain flexibility, which makes the gradual transitions between the domains clearly visible and comparable to available experimental and structural data. In the future, the concept of the fuzzy domains can be used in the coarse‐grained simulations of the domain dynamics in order to account for internal protein flexibility. Proteins 2009. © 2008 Wiley‐Liss, Inc.
The selenoproteome of Clostridium sp. OhILAs: Characterization of anaerobic bacterial selenoprotein methionine sulfoxide reductase A
Authors: Kim Hwa‐Young , Zhang Yan , Lee Byung Cheon , Kim Jae‐Ryong , Gladyshev Vadim N.
Published: September 2008

Selenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In this study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, five of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by 75Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec‐containing MsrA exhibited at least a 20‐fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Crystal structure of the hexamer of human heat shock factor binding protein 1
Authors: Liu Xueqi , Xu Lingfeng , Liu Yiwei , Tong Xiaohang , Zhu Guangyu , Zhang Xuejun C. , Li Xuemei , Rao Zihe
Published: September 2008

Heat shock response (HSR) is a ubiquitous cellular mechanism that copes with a variety of stresses. This response is mediated by a family of transcriptional activators, heat shock factors (HSFs), which are under tight regulation. HSF binding protein 1 (HSBP1) is a negative regulator of HSR and is reported to bind specifically with the active trimeric form of HSF1, thus inhibiting its activity. HSBP1 contains heptad‐repeats in the primary sequence and was believed to stay in a trimer form in solution. We report the crystal structure of the trimerization domain of the M30I/L55P mutant of human HSBP1 at 1.8 Å resolution. In this crystal form, the HSBP1 fragment of residues 6–53 forms a continuous, 11‐turn long helix. The helix self‐associates to form a parallel, symmetrical, triple coiled‐coil helix bundle, which further assembles into a dimer of trimers in a head‐to‐head fashion. Solution study confirmed that the wild‐type HSBP1 shares similar biophysical properties with the crystallized variant. Furthermore, we identified Ser31, which buried its polar side chain in the hydrophobic interior of the helix bundle, as a stability weak‐spot. Substitution of this residue with Ile increases the melting temperature by 24°C, implicating that this conserved serine residue is maintained at position 31 for functional purposes. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Molecular dynamics study of chemically engineered green fluorescent protein mutants: Comparison of intramolecular fluorescence resonance energy transfer rate
Authors: Mitchell Felicity L. , Frank Filipp , Marks Gabriel E. , Suzuki Miho , Douglas Kenneth T. , Bryce Richard A.
Published: September 2008

Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N‐terminal or C‐terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, κ2. In agreement with experiment, the construct with the C‐terminally attached dye was predicted to have higher energy transfer rate than observed for the N‐terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, κ2, deviates from the commonly assumed value, the implications of which are also considered. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange
Authors: Mohan P. M. Krishna , Chakraborty Swagata , Hosur Ramakrishna V.
Published: September 2008

Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native‐state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel β‐sheets (β4 and β5) that form the hydrophobic core of the protein and the α2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant‐dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Theoretical modeling of the O‐intermediate structure of bacteriorhodopsin
Authors: Watanabe Hiroshi C. , Ishikura Takakazu , Yamato Takahisa
Published: September 2008

Bacteriorhodopsin is a prototype of efficient molecular machinery functioning as a light‐activated proton pump. Among the five distinct intermediates (K, L, M, N, and O) of the photocycle, there is less structural information on the later stages compared with the early intermediates. Here, we report the structural modeling of the O‐intermediate for which the determination of experimental structure remains difficult. Hypothetical conformational change of the molecule from the light‐adapted state to the O‐intermediate state was simulated by gradually changing the protonation state of two residues. To achieve accurate molecular modeling, we carefully constructed a realistic system of the native purple membrane. The modeled structure of the O‐intermediate has some implications about proton transfer in the later stages of the photocycle and the structural response of bacteriorhodopsin to the inner charge distribution. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Protonate3D: Assignment of ionization states and hydrogen coordinates to macromolecular structures
Authors: Labute Paul
Published: September 2008

A new method, called Protonate3D, is presented for the automated prediction of hydrogen coordinates given the 3D coordinates of the heavy atoms of a macromolecular structure. Protonate3D considers side‐chain “flip,” rotamer, tautomer, and ionization states of all chemical groups, ligands, and solvent, provided suitable templates are available in a parameter file. The energy model includes van der Waals, Coulomb, solvation, rotamer, tautomer, and titration effects. The results of computational validation experiments suggest that Protonate3D can accurately predict the location of hydrogen atoms in macromolecular structures. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Structural analysis of fish versus mammalian hemoglobins: Effect of the heme pocket environment on autooxidation and hemin loss
Authors: Aranda Roman , Cai He , Worley Chad E. , Levin Elena J. , Li Rong , Olson John S. , Phillips George N. , Richards Mark P.
Published: September 2008

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin ∼50‐ to 100‐fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian α and β chains. The larger IleE11 side chain sterically hinders bound O2 and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme‐7‐propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme‐6 propionate in α subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near β CD3 in the perch Hb (∼8 Å) compared with trout IV Hb (∼6 Å) which in turn is significantly higher than that in bovine Hb (∼4 Å) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E‐helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Computational insights into the interaction of the anthrax lethal factor with the N‐terminal region of its substrates
Authors: Joshi Manali , Ebalunode Jerry Osagie , Briggs James M.
Published: September 2008

The anthrax lethal factor is a zinc metalloprotease toxin secreted by Bacillus anthracis which cleaves at the N‐terminal region of six mitogen activated protein kinase kinases (MEKs) in the cell. Additionally, it is known to cleave a nine residue peptide “LF10,” 50‐fold more efficiently than nine residues of MEK1. There is very little sequence similarity between the MEK N‐termini, thus, it is unclear how the lethal factor can accommodate and cleave the diverse N‐termini of the MEKs and whether there is a hierarchy in this interaction, as there is between LF10 and MEK1. To investigate this problem, we carried out multiple molecular dynamics simulations of the lethal factor with nine residues of each of the substrates. Our simulations reveal that like LF10, certain MEK substrates have residue compositions that favor β‐sheet formation with the lethal factor over others. The formation of this secondary structure maintains a catalytic conformation. Binding energetics using the MM‐PBSA method was used to rank‐order the substrates for their affinity to LF (KM). On the basis of the results, we conclude that the LF does not equally accommodate the MEK substrates and further predict that there will be differences between rates of cleavage among the nine residue MEK N‐termini. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Identification of selectivity‐determining residues in cytochrome P450 monooxygenases: A systematic analysis of the substrate recognition site 5
Authors: Seifert Alexander , Pleiss Jürgen
Published: September 2008

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity‐ and specificity‐determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme‐interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme‐interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme‐interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty‐seven percent of all CYPs are predicted to contain a second selectivity‐determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR‐X(7)‐{P}‐x‐P‐[HKR]. Proteins 2009. © 2008 Wiley‐Liss, Inc.
Mechanism for multiple‐substrates recognition of α‐aminoadipate aminotransferase fromThermus thermophilus
Authors: Tomita Takeo , Miyagawa Tomoharu , Miyazaki Takashi , Fushinobu Shinya , Kuzuyama Tomohisa , Nishiyama Makoto
Published: September 2008

α‐Aminoadipate aminotransferase (AAA‐AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2‐oxoadipate to yield α‐aminoadipate in lysine biosynthesis through the α‐aminoadipate pathway inThermus thermophilus. AAA‐AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA‐AT in four forms: with pyridoxal 5′‐phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), withN‐phosphopyridoxyl‐leucine (PPL) (PPL complex), and withN‐phosphopyridoxyl‐α‐aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 Å resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 Å) of the α2 helix and the β1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the α2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the α2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA‐AT can recognize various kinds of substrates using the mobile α2 helix. The crystal structures and site‐directed mutagenesis revealed that intersubunit‐electrostatic interactions contribute to the elevated thermostability of this enzyme. Proteins 2009. © 2008 Wiley‐Liss, Inc.
AU‐rich RNA‐binding induces changes in the quaternary structure of AUH
Authors: Kurimoto Kazuki , Kuwasako Kanako , Sandercock Alan M. , Unzai Satoru , Robinson Carol V. , Muto Yutaka , Yokoyama Shigeyuki
Published: September 2008

The human AU RNA binding protein/enoyl‐Coenzyme A hydratase (AUH) is a 3‐hydroxy‐3‐methylglutaconyl‐CoA dehydratase in the leucine degradation pathway. It also possesses an RNA‐binding activity to AUUU repeats, which involves no known conserved RNA‐binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)24A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes. Proteins 2009. © 2008 Wiley‐Liss, Inc.