bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated sample treatment with the injection of large sample volumes for the determination of contaminants and metabolites in urine
Authors: Rodríguez‐Gonzalo Encarnación , García‐Gómez Diego , Herrero‐Hernández Eliseo , Carabias‐Martínez Rita
Published: June 2010

This work reports the development of a simple and automated method for the quantitative determination of several contaminants (triazine, phenylurea, and phenoxyacid herbicides; carbamate insecticides and industrial chemicals) and their metabolites in human urine with a simplified sample treatment. The method is based on the online coupling of an extraction column with RP LC separation–UV detection; this coupling enabled fast online cleanup of the urine samples, efficiently eliminating matrix components and providing appropriate selectivity for the determination of such compounds. The variables affecting the automated method were optimized: sorbent type, washing solvent and time, and the sample volume injected. The optimized sample treatment reported here allowed the direct injection of large volumes of urine (1500 μL) into the online system as a way to improve the sensitivity of the method; limits of detection in the 1–10 ng/mL range were achieved for an injected volume of 1500 μL of urine, precision being 10% or better at a concentration level of 20 ng/mL. The online configuration proposed has advantages such as automation (all the steps involved in the analysis – injection of the urine, sample cleanup, analyte enrichment, separation and detection – are carried out automatically) with high precision and sensitivity, reducing manual sample manipulation to freezing and sample filtration.
Problems, artifacts and solutions in the INADEQUATE NMR experiment
Authors: Bain Alex D. , Hughes Donald W. , Anand Christopher K. , Nie Zhenghua , Robertson Valerie J.
Published: June 2010

The INADEQUATE experiment can provide unequalled, detailed information about the carbon skeleton of an organic molecule. However, it also has the reputation of requiring unreasonable amounts of sample. Modern spectrometers and probes have mitigated this problem, and it is now possible to get good structural data on a few milligrams of a typical organic small molecule. In this paper, we analyze the experiment step by step in some detail, to show how each part of the sequence can both contribute to maximum overall sensitivity and can lead to artifacts. We illustrate these methods on three molecules: 1‐octanol, the steroid 17α‐ethynylestradiol and the isoquinoline alkaloid β‐hydrastine. In particular, we show that not only is the standard experiment powerful, but also a version tuned to small couplings can contribute vital structural information on long‐range connectivities. If the delay in the spin echo is long, pairs of carbons with small couplings can create significant double‐quantum coherence and show correlations in the spectrum. These are two‐ and three‐bond correlations in a carbon chain or through a heteroatom in the molecule. All these mean that INADEQUATE can play a viable and important role in routine organic structure determination. Copyright © 2010 John Wiley & Sons, Ltd.
Clinical and electrophysiological features of the 2007 Guillain–Barré syndrome epidemic in Northeast China
Authors: Ye Yuqin , Zhu Dan , Wang Keren , Wu Jiang , Feng Jiachun , Ma Dihui , Xing Yingqi , Jiang Xinmei
Published: June 2010

Guillain–Barré syndrome (GBS) generally presents sporadically. Epidemics of GBS are unusual. We reviewed the medical records of 26 patients hospitalized for GBS during the 2007 GBS epidemic in northeast China. The objective was to determine whether there were clinical and electrophysiological characteristics. All patients had drunk unboiled water, and the illness was preceded by diarrhea in 19 (73%) patients. Only 1 patient had a Campylobacter jejuni infection, whereas 14 (54%) patients exhibited features of acute motor axonal neuropathy (AMAN). The most common electrophysiological findings in early GBS included decreased compound muscle action potential (CMAP) amplitude (62%), abnormal F waves (73%), and abnormal H reflexes (62%). This epidemic of GBS appears to have been associated with consumption of contaminated water. The main subtype was AMAN, which was associated with a longer duration of illness and a worse prognosis. Electrodiagnostic evaluations are helpful for diagnosis in the primary stages of GBS. Muscle Nerve, 2010
Inverse correlation between VEGF and soluble VEGF receptor 2 in POEMS with AIDP responsive to intravenous immunoglobulin
Authors: Terracciano Chiara , Fiore Stefania , Doldo Elena , Manzari Vittorio , Marfia Girolama Alessandra , Bernardi Giorgio , Massa Roberto , Albonici Loredana
Published: June 2010

POEMS (polyneuropathy, organomegaly, endocrinopathy, M‐band, and skin changes) syndrome is characterized by chronic progressive polyneuropathy and plasma‐cell dyscrasia. A major diagnostic criterion of POEMS is elevation of circulating vascular endothelial growth factor (VEGF), which is believed to play a pathogenic role in this disease. We report a case of POEMS that presented as relapsing acute inflammatory demyelinating polyneuropathy, in which complete remission after intravenous immunoglobulin (IVIg) treatment was unexpectedly observed. At clinical nadir, the VEGF level was 30‐fold higher, and the soluble form of VEGF receptor 2 (sVEGFR2), which acts as a decoy for VEGF, was 2.7‐fold lower than normal. These changes combined might contribute to the pathogenesis of POEMS, inducing vascular permeability and tissue edema. At 9‐month follow‐up, during clinical remission, VEGF and sVEGFR2 were near normal values. sVEGFR2 reduction is a new finding in POEMS. IVIg treatment may benefit POEMS patients with acute neuropathy by downgrading VEGF release induced by inflammatory cytokines. Muscle Nerve, 2010
Chemotherapy and interferon‐α treatment of Erdheim–chester disease
Authors: Jeon In‐sang , Lee Sang Seon , Lee Min Kyung
Published: June 2010

Erdheim–Chester disease (ECD) is a rare non‐Langerhans cell histiocytosis of an unknown origin. The prognosis of ECD is variable, and it mainly depends on the involved anatomic sites. The treatment modalities have not been standardized. Interferon‐α (IFN) has been reported to be effective in the management of ECD. We report here on an uncommon case with ECD in a 17‐year‐old female who had multiple lesions in the whole body and she was treated with chemotherapy and IFN. She has remained disease‐free for 2 years after the completion of treatment. Pediatr Blood Cancer. 2010;55:745–747. © 2010 Wiley‐Liss, Inc.
The effect of parental mental health on proxy reports of health‐related quality of life in children with sickle cell disease
Authors: Panepinto Julie A. , Hoffmann Raymond G. , Pajewski Nicholas M.
Published: June 2010

Background

The objectives of this study were to evaluate factors that influence agreement between parent‐proxy and child self‐report of health‐related quality of life (HRQL) in sickle cell disease. We hypothesized that the mental health of the parent, parental HRQL and child characteristics would affect agreement.

Procedure

In a cross‐sectional study of children with sickle cell disease, HRQL of the child and the parent's HRQL and mental health were assessed. The effect of parent and child characteristics on agreement between parent‐proxy and child self‐report of HRQL were determined.

Results

Rates of agreement between parent‐proxy and child self‐report of HRQL ranged between 42% and 49%. Parents with increased symptoms of distress had an increased odds of reporting a worse physical (Odds Ratio (OR) 1.12) and psychosocial HRQL (OR 1.10) compared to the child's self‐report. Severe sickle cell disease was associated with an increased odds of the parent reporting the child's physical HRQL was worse, (OR 4.68) compared to the child's self‐report.

Conclusions

Greater symptoms of distress in the parent are associated with worse parent‐proxy report of the child's HRQL. Severe sickle cell disease is associated with greater disagreement between parent‐proxy and child self‐report of HRQL. These findings broaden our understanding of factors that influence proxy‐reporting of a child's HRQL. Pediatr Blood Cancer. 2010;55:714–721. © 2010 Wiley‐Liss, Inc.
Effect of rubber matrix type on the morphology and reinforcement effects in carbon black‐nanoclay hybrid composites—A comparative assessment
Authors: Praveen S. , Chattopadhyay P.K. , Jayendran S. , Chakraborty B.C. , Chattopadhyay S.
Published: June 2010

Carbon black (B)–nanoclay (NC) hybrid composites, based on millable polyurethane and brominated isoprene isobutylene rubber (BIIR), were prepared. The carbon black loading was fixed at 20 phr and organically modified sodium montmorillonite clay loading varied from 5 to 20 phr in each rubber compounds. The nanocomposites were prepared in laboratory by mixing two‐roll mill. The state of dispersion of the layered silicate was studied by X‐ray diffraction (XRD) and transmission electron microscopy (TEM). XRD and TEM results indicated intercalation of PU and BIIR into the silicate interlayers, followed by exfoliation of the silicate layers into the elastomer matrices. However, the level of intercalation and exfoliation varied considerably with the type of elastomer. The reinforcing effects obtained were found to depend strongly on the extent and degree of the dispersion of the carbon black and silicate layers into the rubber matrices. Mechanical and dynamic mechanical properties were evaluated for each composite. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers
Replication and comparison of concave and convex microlens arrays of light guide plate for liquid crystal display in injection molding
Authors: Kim Jong Sun , Kim Dong Sung , Kang Jeong Jin , Kim Jong Deok , Hwang Chul Jin
Published: June 2010

A back light unit (BLU) is a key module of a thin‐film transistor liquid crystal display (TFT‐LCD), frequently utilized in various mobile displays. In this study, we experimentally characterize transcription and optical properties of concave and convex microlens arrays (MLAs) of light guide plate (LGP) fabricated by injection molding with polycarbonate as a LGP substrate material. Nickel mold inserts were manufactured by electroforming on the MLA, which was fabricated by the thermal reflow of photoresist microstructures patterned by UV‐photolithography. For the case of convex microlens, the height of replicated microlens was less than that of the mold insert while maintaining almost the same microlens diameter of the mold insert as the location of the microlens is far from the gate. In contrast, for the concave microlens, the diameter of replicated microlens was larger than that of mold insert, while showing almost the same microlens height as the mold insert. From the optical examination of replicated convex and concave MLAs, it was found that a higher luminance of the LGP was achieved by the concave MLAs compared to the convex MLAs with the same number density of microlenses (about 30% enhancement in this case) due to the utilization of a larger amount of light provided by the light sources. POLYM. ENG. SCI., 50:1696–1704, 2010. © 2010 Society of Plastics Engineers
Mehr Licht! Femtosekunden‐Quantenoptik mit Festkörper‐Nanostrukturen
Authors: Bratschitsch Rudolf , Leitenstorfer Alfred
Published: June 2010

Halbleiter‐Quantenpunkte sind vielversprechende Systeme auf Festkörperbasis für quantenoptische Experimente. Mit Femtosekunden‐Lichtimpulsen zeigen wir, wie man dieses Einzelelektronen‐System auf der molekularen Zeitskala präpariert, manipuliert und ausliest. Zudem stellen wir zwei Konzepte vor, Licht effizient an solche nanoskopischen Objekte zu koppeln. Eine Möglichkeit hierfür sind optische Mikroresonatoren, in denen die Quantenpunkte sitzen. Alternativ untersuchen wir metallische Nanoantennen, die sichtbares Licht in einer Lücke zwischen ihren beiden Antennenteilen konzentrieren können. Solche Konzepte eröffnen neue Wege in eine Einzelphoton‐Femtosekunden‐Physik.
Die energiereichsten kosmischen Teilchen. Ergebnisse des Pierre‐Auger‐Observatoriums
Authors: Blümer Johannes
Published: June 2010

Seit Mitte 2008 ist das Pierre‐Auger‐Observatorium in Argentinien zur Beobachtung der kosmischen Strahlung bei höchsten Energien vollständig ausgebaut. Es hat erstmals ein solide kalibriertes Energiespektrum, eine Messung der Schauereigenschaften und Primärmassen sowie eine anisotrope Verteilung der Ankunftsrichtungen der Teilchen geliefert. So gibt es statistisch signifikante Hinweise darauf, dass Teilchen mit Energien von mehr als 1019 eV mit den Positionen von aktiven Galaxien korrelieren. Dennoch bleiben Fragen offen, die mit dem zukünftigen Observatorium Auger‐Nord beantwortet werden sollen.
Cleavages in stable tetrahedral intermediates' bonds and the role of water
Authors: Bethencourt Loengrid , Vivas Daniel , Núñez Oswaldo
Published: June 2010

Using two‐dimensional energy diagrams, results on the formation and cleavage of detectable tetrahedral intermediates are used to predict the position of transition states involved in the cleavages of unstable and stable tetrahedral intermediates (STI). A late transition state (anti‐Hammond) for the former and synchronous cleavage for the latter are predicted and supported with experimental results. For instance, in the case of STI, intrinsic leaving abilities in the order CO > CNHCO ≫ CNH were experimentally observed, confirming the departure of these groups without full heteroatom protonation. The STI designed herein mimics serine proteases enzymes due to the thermoneutral relationship between the enzyme substrate (ES) and enzyme‐acyl tetrahedral intermediate (EATI) analogs. In the STI models, a dramatic change in pKa is observed when changing the lactam ring size due to the strain in the 3‐fused‐ring system of the STI‐H2O complex formed and exo‐anomeric effect. A plot of ΔSo (STI formation) versus pKa provides evidence for the STI–water interaction. Since only one water molecule is required to produce the effect it is also detected when using ‘pure’ CDCl3 as the solvent and therefore may also play a role in the active sites of serine proteases. Copyright © 2010 John Wiley & Sons, Ltd.
Poly[N‐isopropyl acrylamide]‐co‐polyurethane copolymers for controlled release of urea
Authors: Mathews Anu Stella , Narine Suresh
Published: July 2010

A novel block copolymer of poly[N‐isopropyl acrylamide]‐co‐polyurethane was designed, synthesized, and applied as controlled release fertilizer coating. Structural confirmation of the copolymer was performed using FTIR and 1H‐NMR spectra and elemental analysis. The coating process consists essentially of immersing urea granules in molten polymer and removing the coated urea from the melt by centrifugal action. The morphology of the coated urea was studied using scanning electron microscope (SEM). The polymer coat of the urea granules was found to swell in water forming pores and enabling the release of urea. The urea released from the granule, monitored using a mass spectroscopy technique, was found to be governed by pH of the aqueous medium. The study anticipates development of a beneficiary fertilizer coat in terms of improving controlled release over a period of time which can be tailored by soil temperature, pH and moisture. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3236–3243, 2010
Poly(lactic acid) brushes grow longer at lower temperatures
Authors: Xu Lebo , Gorman Christopher B.
Published: July 2010

Poly(lactic acid) (PLA) brushes prepared by the ring opening of lactide were thicker when polymerized at a lower temperature (25 °C) than was typically used for polymerization in solution. The molecular weight of solution polymerized lactide was also higher when lactide was polymerized at 25 °C compared with polymerization at higher temperatures. However, the yield of PLA was low at this temperature. These results highlight the different requirements for solution polymerization and brush growth. In the former case, both percentage of conversion and molecular weight are important considerations. In the latter case, however, percentage of conversion is unimportant as a brush represents a very small amount of polymer. It was also shown during the course of these studies that the native hydroxy groups on silicon substrates and silanols in solution were equally good initiators when compared with hydroxy terminated self‐assembled monolayers on gold and alcohols, respectively. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3362–3367, 2010
The spectrophotometric study of a fluorescence‐enhanced inclusion complex threaded with β‐cyclodextrin: Synthesis and characterization
Authors: Chiu Yi‐Hong , Liu Jui‐Hsiang
Published: July 2010

A novel achiral monomer end‐capped with a phenyl‐[1,3,4]oxadiazolyl group and threaded through β‐cyclodextrin was synthesized to investigate the host‐guest interactions in the inclusion complex. 1H NMR studies revealed that one or two cyclodextrin molecules were threaded onto the synthesized achiral monomer, leading to the formation of a fibrous construction of self‐assembled inclusion complexes. The formation of a self‐assembled inclusion complex was identified using SEM and TEM. The highly ordered alignment of self‐assembled supramolecules was confirmed using polarized optical microscopy. We demonstrate an easy process for the fabrication of nano‐structured self‐assembled inclusion complexes in pyridine/ethanol (1 mL/10 mL) as well as the enhancement of photo‐induced fluorescence via monomers end‐capped with a phenyl‐[1,3,4]oxadiazolyl moiety threaded with β‐cyclodextrins. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3368–3374, 2010
Selective single monomer addition in living cationic polymerization: Sequential double end‐functionalization in combination with capping agent
Authors: Ida Shohei , Terashima Takaya , Ouchi Makoto , Sawamoto Mitsuo
Published: June 2010

Amine‐functionalized and amine‐carboxylate double‐functionalized polymers (I and II, respectively) have been synthesized by a selective single addition of a protected 2‐aminoethyl vinyl ether (BocVE) {CH2 = CH[OCH2CH2N(Boc)2]; Boc = t‐butoxycarbonyl} onto a living cationic poly(n‐butyl vinyl ether) [poly(NBVE)] initiated with the SnCl4/n‐Bu4NCl system: (I) ‐(NBVE)n‐ CH2CH(OCH2CH2NH2)‐H; (II) ‐(NBVE)n‐CH2CH(OCH2CH2NH2)‐CH2CO2H. The single addition was examined with a set of alkene monomers less reactive than NBVE, including BocVE, 2‐chloroethyl vinyl ether, 2‐vinyloxyethylphtalimide, and styrene (St). Upon addition of 10 molar excess of these alkenes onto the living ends, only BocVE led to the intended single adduct, and this was attributed to a chelating interaction of the two carboxylate groups in the terminal BocVE unit with the growing poly(NBVE) terminal, thus sterically hampering further propagation. A simple acid‐catalyzed Boc‐deprotection led to the amino‐functionalized version I. Alternatively, an additional quenching the BocVE‐capped living end (the precursor of I) with sodium malonate, followed by double deprotection of the Boc and the malonate groups gave the double‐functionalized version II. The selective addition of a single monomer molecule is thus a new method for addressable or site‐specific introduction of functional groups along polymer chains. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3375–3381, 2010
Photoluminescent energy transfer from poly(phenyleneethynylene)s to near‐infrared emitting fluorophores
Authors: Levine Mindy , Song Inja , Andrew Trisha L. , Kooi Steven E. , Swager Timothy M.
Published: July 2010

Photoluminescent energy transfer was investigated in conjugated polymer‐fluorophore blended thin films. A pentiptycene‐containing poly(phenyleneethynylene) was used as the energy donor, and 13 fluorophores were used as energy acceptors. The efficiency of energy transfer was measured by monitoring both the quenching of the polymer emission and the enhancement of the fluorophore emission. Near‐infrared emitting squaraines and terrylenes were identified as excellent energy acceptors. These results, where a new fluorescent signal occurs in the near‐infrared region on a completely dark background, offer substantial possibilities for designing highly sensitive turn‐on sensors. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3382–3391, 2010
Electron microscopic study of the structure of polymers in solutions
Authors: Fakirov S. , Kargin V. A. , Bakeev N. F.
Published: July 2010

The well‐known and widely used methods of samples preparation for electron microscopic studying cannot be employed for polymer solutions. In the present work two techniques have been proposed which are free of the effects produced due to evaporation of the solvent. Their basic principles are as follows: (1) As a solvent a substance which easily goes into a vitreous state on cooling and has glass temperature higher than the room one is used. (2) The solvent is a substance with a low critical temperature (Te). The solvent is condensed in a glass capillary tube containing the polymer and then the capillary tube is heated in a special furnace in vacuo. When the temperature of the furnace is 20–25°C. higher than Te of the polymer solution the capillary tube is broken. Thus the content is “shot” at the grids which can be examined under the electron microscope. Using the described techniques the following systems were studied: polybutene‐1 in colophony, polybutene‐1 in propane, and atactic polypropylene in propane (at concentrations ranging from 0.07 to 3% by weight). Thus, by means of direct electron microscopy, it has been found that in polymer solutions the microscopic structuring of the polymer takes place long before lamination. The appearance of the ordered super‐molecular structures strongly depends on the concentration of the polymer solution.
Mechanism of the polymerization of vinyl chloride and chloroprene in the presence of transition‐metal complexes
Authors: Yamazaki Noboru , Kambara Shu
Published: July 2010

To clarify the polymerization mechanism of vinyl chloride (VCl) and chloroprene (CP) with titanium tetrabutoxide‐aluminum alkylchlorides some work was carried out. Carbon tetrachloride surprisingly enhanced polymerization without decreasing the molecular weight, whereas carbon tetrabromide transferred the polymer chain. The monomer reactivity ratios in the copolymerization of VCl with vinylidene chloride (VdCl), rVCl = 0.30 and rVdCl = 0.90, differed from those in free‐radical polymerizations. In the polymerization of perdeuterovinyl chloride an ethyl radical originating from AlCl(C2H5)2 was found to be incorporated into the polymer. Polymerization of chloroprene with the same catalyst in carbon tetrachloride was kinetically investigated, and a square‐root dependence of the catalyst concentration on the rate was seen. With vanadyl or cobalt acetylacetonate polymers of identical microstructure were obtained. Copolymerization with butadiene and styrene revealed the same behavior as that in free‐radical polymerization. It is suggested that in the polymerization with this catalyst an ionic or coordinated mechanism is included besides the usual radical mechanism.
Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: A model of the GVP‐ssDNA complex
Authors: Guan Yue , Zhang Hong , Wang Andrew H.‐J.
Published: June 2010

The crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, f1, fd) has been solved for the wild‐type and two mutant (Y41F and Y41H) proteins at high resolution. The Y41H mutant crystal structure revealed crystal packing interactions, which suggested a plausible scheme for constructing the polymeric protein shell of the GVP‐single‐stranded DNA (ssDNA) complex (Guan Y, et al., 1994, Biochemistry 33:7768–7778). The electrostatic potentials of the isolated and the cooperatively formed protein shell have been calculated using the program GRASP and they revealed a highly asymmetric pattern of the electrostatic charge distribution. The inner surface of the putative DNA‐binding channel is positively charged, whereas the opposite outer surface is nearly neutral. The electrostatic calculation further demonstrated that the formation of the helical protein shell enhanced the asymmetry of the electrostatic distribution. A model of the GVP‐ssDNA complex with the n = 4 DNA‐binding mode could be built with only minor conformational perturbation to the GVP protein shell. The model is consistent with existing biochemical and biophysical data and provides clues to the properties of GVP, including the high cooperativity of the protein binding to ssDNA. The two antiparallel ssDNA strands form a helical ribbon with the sugar‐phosphate backbones at the middle and the bases pointing away from each other. The bases are stacked and the Phe 73 residue is intercalated between two bases. The optimum binding to a tetranucleotide unit requires the participation of four GVP dimers, which may explain the cooperativity of the GVP binding to DNA.
Protein destabilization by electrostatic repulsions in the two‐stranded α‐helical coiled‐coil/leucine zipper
Authors: Kohn Wayne D. , Kay Cyril M. , Hodges Robert S.
Published: June 2010

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two‐stranded α‐helical coiled‐coils as a model system. The native coiled‐coil consists of two identical 35‐residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled‐coil structure at 20 °C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled‐coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled‐coils contained potential i to i' +5 Glu‐Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu‐Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled‐coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 °C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu‐Glu repulsion in the E2(20,22) coiled‐coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' +5 interchain Glu‐Glu repulsions, which destabilize the coiled‐coil by 0.45 kcal/mol each. At pH 3 the coiled‐coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed‐phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled‐coil interface leading to higher coiled‐coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled‐coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled‐coil stability in the presence of GdnHCl.
A proposed structural model of domain 1 of fasciclin III neural cell adhesion protein based on an inverse folding algorithm
Authors: Castonguay Laurie A. , Snow Peter M. , Fetrow Jacquelyn S. , Bryant Stephen H.
Published: June 2010

Fasciclin III is an integral membrane protein expressed on a subset of axons in the developing Drosophila nervous system. It consists of an intracellular domain, a transmembrane region, and an extracellular region composed of three domains, each predicted to form an immunoglobulin‐like fold. The most N‐terminal of these domains is expected to be important in mediating cell‐cell recognition events during nervous system development. To learn more about the structure/function relationships in this cellular recognition molecule, a model structure of this domain was built. A sequence‐to‐structure alignment algorithm was used to align the protein sequence of the fasciclin III first domain to the immunoglobulin McPC603 structure. Based on this alignment, a model of the domain was built using standard homology modeling techniques. Side‐chain conformations were automatically modeled using a rotamer search algorithm and the model was minimized to relax atomic overlaps. The resulting model is compact and has chemical characteristics consistent with related globular protein structures. This model is a de novo test of the sequence‐to‐structure alignment algorithm and is currently being used as the basis for mutagenesis experiments to discern the parts of the fasciclin III protein that are necessary for homophilic molecular recognition in the developing Drosophila nervous system.
Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A)
Authors: Mcmullen Brad A. , Fujikawa Kazuo , Davie Earl W. , Hedner Ulla , Ezban Mirella
Published: June 2010

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.
Site‐specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion
Authors: Schindler Patrick A. , Settineri Christine A. , Collet Xavier , Fielding Christopher J. , Burlingame Alma L.
Published: June 2010

Site‐specific structural characterization of the glycosylation of human lecithinrcholesterol acyltransferase (LCAT) was carried out using microbore reversed‐phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate‐specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected TV‐linked glycopeptides of LCAT, a di‐O‐linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N‐linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O‐linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O‐linked glycopeptide, each of which contain sialylated galactoseβ13N‐acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N‐linked glycosylation sites at Asn43 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 7766:301–304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.
Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein
Authors: Smith Stanley V. , Correia John J. , Case Steven T.
Published: June 2010

We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino‐terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.
Aminolevulinate synthase: Lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis
Authors: Vajapey Usha , Hafez Osama , Hunter Gregory A. , Barber Michael J. , Ferreira Gloria C.
Published: June 2010

5‐Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine‐313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5′‐phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959–1965). Here we report on the effect of replacement of aminolevulinate synthase lysine‐313 by alanine, histidine, and glycine, using site‐directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild‐type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5′‐phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5′‐phosphate co‐factor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase‐catalyzed reaction. In contrast, lysine‐313 is an essential catalytic residue, because the K313‐directed mutant enzymes have no measurable activity. In summary, site‐directed mutagenesis of the aminolevulinate synthase active‐site lysine‐313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5′‐phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine‐313 has a functional role in catalysis.
Characterization of the N‐terminal half‐saturated state of calbindin D9k: NMR studies of the N56A mutant
Authors: Wimberly Brian , Chazin Walter J. , Thulin Eva
Published: June 2010

Calbindin D9k is a small EF‐hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N‐terminal site (I) is investigated by NMR experiments on the half‐saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038–1044). Analysis of calcium‐induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N‐terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium‐loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild‐type protein, a model for the complementary half‐saturated state with an ion bound in the C‐terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half‐saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF‐hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.
A three‐dimensional model of aromatase cytochrome P450
Authors: Graham‐Lorence Sandra , Peterson Julian A. , Amarneh Bilal , Simpson Evan R. , White Ronald E.
Published: June 2010

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane‐bound proteins, they have resisted structure‐function analysis by means of X‐ray crystallographic methods. Among P450‐catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three‐dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox‐partner binding, which require examination of the entire molecule (excepting the putative membrane‐spanning region). This model of P450arom was built based on a “core structure” identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM‐P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane‐associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redoxpartner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a “threonine switch” determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.
pH dependence of the stability of barstar to chemical and thermal denaturation
Authors: Khurana Ritu , Hate Anita T. , Nath Utpal , Udgaonkar Jayant B.
Published: June 2010

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far‐UV and near‐UV CD, and absorbance as probes. Both GdnHCl‐induced and urea‐induced denaturation studies at pH 7 show that barstar unfolds through a two‐state F ⇌ U mechanism and yields identical values for ΔGU the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 ± 0.5 kcal·mol−1 at 25 °C. Thermal denaturation of barstar also follows a two‐state F ⇌ U unfolding transition at pH 7, and the value of ΔGU at 25 °C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2–10, because barstar does not unfold through a two‐state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2–3, where barstar exists in a molten globule‐like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild‐type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 ± 0.2, is not observed in H17Q, a His 17 ± Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 ± 0.2, is not detected in BSCCAA, the Cys 40 Cys 82 → Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 ± 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies. In the pH range where both chemical denaturation and thermal denaturation studies were possible, both Cm and Tm, the midpoint of a thermal denaturation curve displays similar trends. Very high pH (pH 12) is shown to completely unfold the protein in a fully reversible manner.
Crystallization and preliminary X‐ray crystallographic studies on Type III antifreeze protein
Authors: Jia Zongchao , Deluca Carl I. , Davies Peter L.
Published: June 2010

Type III antifreeze protein, more specifically the recombinant QAE‐Sephadex‐binding isoform, has been crystallized in 50–55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0–4.5. The resultant crystals belong to the ortho‐rhombic space group P212121 with a = 32.60 Å, b = 39.00 Å, and c = 46.57 Å and diffract to at least 1.7 Å. A set of 1.7‐Å native data has been collected, with completeness 93.4% and Rsvm of 0.069. Initial screening for heavy‐atom derivatives has yielded a Pt‐bound derivative.
Generating stereochemically acceptable protein pathways
Authors: Farrell Daniel W. , Speranskiy Kirill , Thorpe M. F.
Published: June 2010

We describe a new method for rapidly generating stereochemically acceptable pathways in proteins. The method, called geometric targeting, is publicly available at the webserver http://pathways.asu.edu, and includes tools for visualization of the pathway and creating movie files for use in presentations. The user submits an initial structure and a target structure, and a pathway between the two input states is generated automatically. Besides visualization, the structural quality of the pathways makes them useful as input pathways into pathway refinement techniques and further computations. The approach in geometric targeting is to gradually change the system's RMSD relative to the target structure while enforcing a set of geometric constraints. The generated pathways are not minimum free energy pathways, but they are geometrically plausible pathways that maintain good covalent bond distances and angles, keep backbone dihedral angles in allowed Ramachandran regions, avoid eclipsed side‐chain torsion angles, avoid non‐bonded overlap, and maintain a set of hydrogen bonds and hydrophobic contacts. Resulting pathways for over 20 proteins featuring a wide variety of conformational changes are reported here, including the very large GroEL complex. Proteins 2010. © 2010 Wiley‐Liss, Inc.
Quantum chemical studies of some rhodanine azosulpha drugs as corrosion inhibitors for mild steel in acidic medium
Authors: Ebenso Eno E. , Arslan Taner , Kandemirli Fatma , Caner Necmettin , Love Ian
Published: July 2010

The density functional theory (DFT) at the B3LYP/6‐31G (d,p) and B3LYP/6‐311G(d,p) basis set levels and ab initio calculations using the HF/6‐31G (d,p) and HF/6‐311G(d,p) methods were performed on four rhodanine azosulpha drugs (namely 5‐sulfadiazineazo‐3‐phenyl‐2‐thioxo‐4‐thiazolidinone, 5‐ sulfamethazineazo‐3‐phenyl‐2‐thioxo‐4‐thiazolidinone, 5‐sulfadimethoxineazo‐3‐phenyl‐2‐thioxo‐ 4‐thiazolidinone, and 5‐sulfamethoxazoleazo‐3‐phenyl‐2‐thioxo‐4‐thiazolidinone) used as corrosion inhibitors for mild steel in acidic medium to determine the relationship between the molecular structure of the rhodanine azosulpha drugs and inhibition efficiency(%IE). The quantum chemical parameters/descriptors, namely, EHOMO (highest occupied molecular orbital energy), ELUMO (lowest unoccupied molecular orbital energy), the energy difference (ΔE) between EHOMO and ELUMO, dipole moment (μ), electron affinity (A), ionization potential (I), the absolute electronegativity (X), absolute hardness (η), softness (σ), polarizability (α), the Mulliken charges, and the fraction of electrons (ΔN) transfer from inhibitors to iron, were calculated and correlated with the experimental %IE. Quantitative structure activity relationship (QSAR) approach has been used, and a composite index of some quantum chemical parameters/descriptors was performed to characterize the inhibition performance of the studied molecules. The results showed that the inhibition efficiency (%IE) of the rhodanine azo sulfa drugs studied was closely related to some of the quantum chemical parameters/descriptors but with varying degrees of correlation coefficient (R2). The %IE also increased with the increase in EHOMO and decrease in EHOMO−ELUMO; and the areas containing N atoms are the most possible sites for bonding to the metal iron surface by donating electrons to the metal. The HOMO orbitals consist of 61.73–63.04% double bonded S atom (7(S)), and most of the rest are concentrated on the rhodanine group; so, the rhodanine molecule plays an important role in bonding of sulfa drugs with metal atom in the corrosion process. The calculated/estimated %IE of the compounds studied was found to be close to the experimental %IE. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010