bluuebunny/crossref_metadata_2025 huggingface dataset:
VEGF‐R2 and neuropilin‐1 are involved in VEGF‐A‐induced differentiation of human bone marrow progenitor cells
Authors: Fons Pierre , Herault Jean‐Pascal , Delesque Nathalie , Tuyaret Joēl , Bono Françoise , Herbert Jean‐Marc
Published: April 2004

Tumor growth and metastasis require the generation of new blood vessels, a process known as neo‐angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor‐A (VEGF‐A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF‐receptor tyrosine kinases and about the implication of the VEGF‐R co‐receptor, neuropilin‐1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM‐AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF‐A on BM‐AC133+ cells relies on an early action of VEGF‐A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF‐R2/neuropilin‐1‐dependent signaling pathway. This signaling promotes the differentiation of BM‐AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF‐R2 and/or neuropilin‐1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth. © 2004 Wiley‐Liss, Inc.
Vertex‐disjoint cycles containing specified vertices in a bipartite graph
Authors: Chen Guantao , Enomoto Hikoe , Kawarabayashi Ken‐ichi , Ota Katsuhiro , Lou Dingjun , Saito Akira
Published: April 2004

A minimum degree condition is given for a bipartite graph to contain a 2‐factor each component of which contains a previously specified vertex. © 2004 Wiley Periodicals, Inc. J Graph Theory 46: 145–166, 2004
A Gallai–Edmonds‐type structure theorem for path‐matchings
Authors: Spille Bianca , Szegő László
Published: April 2004

As a generalization of matchings, Cunningham and Geelen introduced the notion of path‐matchings. We give a structure theorem for path‐matchings which generalizes the fundamental Gallai–Edmonds structure theorem for matchings. Our proof is purely combinatorial. © 2004 Wiley Periodicals, Inc. J Graph Theory 46: 93–102, 2004
Regular 4‐critical graphs of even degree
Authors: Dobrynin Andrey A. , Melnikov Leonid S. , Pyatkin Artem V.
Published: April 2004

In 1960, Dirac posed the conjecture that r‐connected 4‐critical graphs exist for every r ≥ 3. In 1989, Erdős conjectured that for every r ≥ 3 there exist r‐regular 4‐critical graphs. In this paper, a technique of constructing r‐regular r‐connected vertex‐transitive 4‐critical graphs for even r ≥ 4 is presented. Such graphs are found for r = 6, 8, 10. © 2004 Wiley Periodicals, Inc. J Graph Theory 46: 103–130, 2004
Chloride transport in the vegetative mycelia of filamentous fungus Trichoderma viride
Authors: Šimkovič M. , Pokorný R. , Hudecová D. , Varečka Ľ.
Published: April 2004

The influx of chlorides into Trichoderma viride vegetative submerged mycelium was measured by means of the radionuclide 36Cl−. It was found that the 36Cl− influx was time‐dependent (the steady‐state was established with t1/2 = 25 min at 25 °C), pH‐dependent (with pH optimum between 4–5.5), temperature‐dependent (at about 15 °C), and concentration‐dependent (KM = 47.6 ± 4.2 μmol · l−1; Jmax = 11.5 ± 0.7 pmol(Cl−) · min−1 · mgdry mass−1). The 36Cl− influx was inhibited by Br− but not F−, I−, SO42−, HPO32− and HCO3−. The presence of vanadate (P‐type ATPase inhibitor) moderately stimulated the 36Cl− influx but the presence valinomycin (electrogenic K+ ionophore), salicylate (known to release Ca2+ from Trichoderma viride internal stores) were without effect on the 36Cl− influx. The results suggest that the 36Cl− influx is mediated by a carrier and that the transport is electroneutral, probably Cl−/OH− antiport. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Temporal variation in muscle fibre area, gaping, texture, colour and collagen in triploid and diploid Atlantic salmon (Salmo salar L)
Authors: Bjørnevik Marit , Espe Marit , Beattie Chris , Nortvedt Ragnar , Kiessling Anders
Published: April 2004

Diploid and triploid Atlantic salmon were reared for 32 months in seawater, from October 1998 to January 2000. During this period of time, four samplings were taken to study differences in quality traits and chemical components in the flesh between diploid and triploid Atlantic salmon. Season was found to be the dominant factor explaining the variation in flesh quality traits in both triploid and diploid fish. Ploidy affected the majority of investigated variables while body size had lesser impact. Triploid Atlantic salmon had fewer small muscle fibres and up to 23% larger mean cross‐sectional muscle fibre area than diploids. Triploids also displayed more gaping, softer fillet texture, lower post mortem end pH, darker (L value) and redder (a value) flesh colour, and more soluble and less insoluble collagen compared with diploid fish. No effect of ploidy was found on crude chemical composition. Furthermore, a negative relationship was found between gaping score and muscle fibre area, and a weak positive correlation was found between fibre density and texture firmness. However, when body size and sampling time was included in the statistical model, this relationship between gaping and fibre area became very weak, and the relationship between texture and fibre area was completely negated. This suggests that intra‐species variation in both texture and gaping is more related to season and body size than to average muscle fibre area size. Copyright © 2004 Society of Chemical Industry
Sensitivity enhancement of chlorinated phenols by continuous flow liquid membrane extraction followed by capillary electrophoresis
Authors: Zhou Qingxiang , Liu Jingfu , Jiang Guibin , Liu Guoguang , Cai Yaqi
Published: April 2004

This paper describes a new analytical system, based on the combination of continuous flow liquid membrane extraction (CFLME) enrichment and capillary electrophoresis (CE) separation, for analysis of chlorinated phenols in water samples. Five chlorinated phenols including 3‐chlorophenol (3CP), 4‐chlorophenol (4CP) 2,4‐dichlorophenol (DCP), 2,4,6‐trichlorophenol (TCP), and pentachlorophenol (PCP) were separated by CE with Tris/sodium dihydrogen phosphate solution containing methanol 1% (v/v) as the run buffer. CFLME related parameters were investigated and optimal enrichment was obtained by using 0.3 mol L–1 Tris as acceptor and with a sample pH 5.0, a sample flow rate of 4.0 mL min–1, and an enrichment sample volume of 150 mL. The detection limit (S/N = 3) was 6.9, 1.0, and 1.7 ng mL–1 for DCP, PCP, and TCP, respectively. The reproducibility (RSD%, n = 6) was 5.7 for DCP, 2.5 for PCP, and 2.8% for TCP (n = 6). The proposed method was applied to the determination of chlorinated phenols in spiked water samples with relatively satisfactory recoveries.
Sequential injection chromatographic determination of paracetamol, caffeine, and acetylsalicylic acid in pharmaceutical tablets
Authors: Šatínský Dalibor , Neto Isabel , Solich Petr , Sklenářová Hana , Conceição M. , Montenegro B. S. M. , Araújo Alberto N.
Published: April 2004

In this contribution, a new separation method for simultaneous determination of paracetamol, caffeine, acetylsalicylic acid, and internal standard benzoic acid was developed based on a novel reversed‐phase sequential injection chromatography (SIC) technique with UV detection. A Chromolith® Flash RP‐18e, 25‐4.6 mm column (Merck, Germany) and a FIAlab® 3000 system (USA) with an 8‐port selection valve and a 5 mL syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile‐(0.01 M) phosphate buffer (10 : 90, v/v) pH 4.05, flow rate 0.6 mL min–1. UV detection was at 210 and 230 nm. The validation parameters showed good results: linearity (r >0.999) for all compounds, detection limits in the range 0.3–0.8 μg mL–1, repeatability (RSD) of peak heights between runs in the range 1.10–4.30% at three concentration levels and intra‐day repeatability of the retention times in the range 0.28–0.43%. The analysis time was < 6 min. The method was found to be applicable for the routine analysis of the active compounds paracetamol, caffeine, and acetylsalicylic acid in pharmaceutical tablets.
Synthesis and Thermal Properties of Epoxidized Vegetable Oil
Authors: Park Soo‐Jin , Jin Fan‐Long , Lee Jae‐Rock
Published: April 2004

Summary: The novel potential epoxy resins, epoxidized soybean oil (ESO) and epoxidized castor oil (ECO), were synthesized and characterized. The cationic polymerization of ESO and ECO with a latent thermal catalyst, N‐benzylpyrazinium hexafluoroantimonate (BPH), was initiated at 80 and 50 °C, respectively. The cured ECO samples showed a higher Tg and lower coefficient of thermal expansion than those of ESO, due to the higher intermolecular interaction in the ECO/BPH system.

Relationships between ESO or ECO conversion and temperature in the polymerization with 1 wt.‐% BPH for 2 h.

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Relationships between ESO or ECO conversion and temperature in the polymerization with 1 wt.‐% BPH for 2 h.
Gelation Rate Modulation of an α‐Cyclodextrin and Poly(ethylene glycol)‐Grafted Hyaluronic Acid Solution System by Inclusion Complexation of a Microphase‐Separated Structure
Authors: Nakama Tsuyoshi , Ooya Tooru , Yui Nobuhiko
Published: April 2004

Summary: The gelation rate of a poly(ethylene glycol)‐grafted hyaluronic acid (PEG‐graft‐HA) solution with adding α‐CD was investigated in term of the microphase separation between the grafted PEG and HA. The gelation rate of PEG‐graft‐HA exhibiting the microphase‐separated structure was two times higher than that of PEG‐graft‐HA showing a homogeneous miscible state.

The formation of microphases by PEG‐graft‐HA contributes to its rapid gelation upon the addition of α‐CDs.

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The formation of microphases by PEG‐graft‐HA contributes to its rapid gelation upon the addition of α‐CDs.
Synthesis and Characterization of Cross‐Linked Poly(p‐phenylene ethynylene)s
Authors: Hittinger Eric , Kokil Akshay , Weder Christoph
Published: April 2004

Summary: Conjugated poly(p‐phenylene ethynylene) networks with interesting optoelectronic properties were synthesized by the palladium‐catalyzed polycondensation of 2,5‐diiodo‐4‐[(2‐ethylhexyl)oxy]methoxybenzene, and 1,4‐diethynyl‐2,5‐bis‐(octyloxy)benzene, with 1,2,4‐tribromobenzene as cross‐linker. The cross‐linker concentration was varied and materials with different cross‐link densities were prepared. The materials were processed into films by simultaneous polymerization and shaping. An alternative approach is to synthesize these cross‐linked polymers in the form of spherical particles, which can be processed from dispersions.

Schematic representation of the cross‐linking process.

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Schematic representation of the cross‐linking process.
Eine innovativ im Sol‐Gel‐Prozeß hergestellte, hochporöse Siliziumoxidkeramik zum Knochenersatz – In‐vivo Langzeitergebnisse
Authors: Bienengräber V. , Gerber Th. , Trykova T. , Kundt G. , Henkel K.‐O.
Published: April 2004

Es wurden zwei innovativ im Sol‐Gel‐Verfahren bei 200 °Celsius hergestellte Biokeramikvarianten auf Kalziumphosphatbasis mit Siliziumoxid (SiO2) als Adjuvans im Langzeittierversuch getestet. Bei adulten Miniaturschweinen wurden Defekte kritischer Größe (>5cm3) im Bereich der Mandibula gesetzt. In der Versuchsgruppe I (n=6) wurden sie mit einer biphasischen (60 % Hydroxilapatit [HA] und 40 % ß‐Trikalziumphosphat) und in der Gruppe II (n=6) mit einer monophasischen Variante (100 % HA) der Testkeramik aufgefüllt. Die Gruppe III (n=6) bildete die Leerkontrolle. Nach acht Monaten wurden die Tiere getötet; es erfolgte eine klinische, histologische, morphometrische und REM‐Analyse des Knochens der Defektregion. In beiden Versuchsgruppen waren nach 8 Monaten die Defekte durchgehend ossifiziert sowie die Keramiken nahezu vollständig biodegradiert (Gruppe I: 96,5 % / Gruppe II: 99,2 %). Beide im Sol‐Gel‐Verfahren hergestellten SiO2‐Kalziumphosphatkeramiken wiesen im Tierexperiment neben einer sehr guten Osteokonduktivität ein im Vergleich zu herkömmlichen Biokeramiken günstigeres Resorptionsverhalten auf. Sie erscheinen daher für die Therapie knöcherner Defekte beim Menschen sehr geeignet. Eine klinische Studie ist angelaufen.
Disabling repetitive behaviors in Parkinson's disease
Authors: Kurlan Roger
Published: April 2004

We report on 6 patients with Parkinson's disease who developed severe repetitive behavior, 2 with pathological gambling and 4 with cleaning, rearranging, and ordering rituals. The onset of repetitive behaviors was not associated with changes in antiparkinsonian medications, the behavior did not improve after reductions in dopaminergic therapy, and thus they seem to be part of the underlying illness. The disabling cleaning/rearranging/ordering behaviors had associated features of hypomania, occurred exclusively during motor on periods, and often occurred nocturnally. The repetitive behavior responded poorly to serotonin reuptake inhibitors. Certain atypical antipsychotics may be of benefit. © 2003 Movement Disorder Society
Complex mechanisms of sensory tricks in cervical dystonia
Authors: Schramm Axel , Reiners Karlheinz , Naumann Markus
Published: April 2004

Muscle activities in 26 patients with predominantly rotational torticollis were quantified using surface electromyography. In the subgroup of 19 patients with an effective sensory trick, different modes and bilateral locations of trick application led to significant (P < 0.002) reduction of electromyographic (EMG) activity. A strong correlation was found between the efficacy of trick manoeuvres and the starting head position. Trick application in a neutral or even contralateral position was most effective while no reduction of muscle activity during trick application at the maximum dystonic head position was found (P < 0.001). We propose a two‐phase model: First, normalisation of head posture is obtained by counterpressure or volitional antagonistic muscle activity. In a second step, this position can be stabilised using sensory tricks challenging central adaption of distorted sensorimotor integration. © 2003 Movement Disorder Society
Wireless real‐time electronic data capture for self‐assessment of motor function and quality of life in Parkinson's disease
Authors: Nyholm Dag , Kowalski Jan , Aquilonius Sten‐Magnus
Published: April 2004

Frequent assessment of the symptoms in Parkinson's disease (PD) is important in both clinical and experimental settings, especially when motor fluctuations are present. Patient diaries are increasingly used in studies, allowing patients to stay in their home environments. However, traditional paper diaries may not reflect reality because of a lack in compliance or retrospective data entries. This study presents a comparison between paper diaries and a new method, real‐time data capture with a hand‐held computer (electronic diary). Twenty patients with PD diagnosed at least 5 years previously were randomly assigned to use either a paper diary or an electronic diary on 8 days during 1 month. Questions were answered every 2 hours over a 12‐hour period on each day. Median compliance was 88% with the electronic diary and 98% with the paper diary, although strict compliance to the scheduled times by patients using the paper diary was 78%. Neither age nor earlier experience with computers affected the patient's ability to use the electronic diary. Electronic diaries can be used for self‐assessment of PD symptoms. The real‐time feature provides fast access to clean data with knowledge of true compliance. © 2003 Movement Disorder Society
Progressive supranuclear palsy syndrome induced by clebopride
Authors: Campdelacreu Jaume , Kumru Hatice , Tolosa Eduard , Valls‐Solé Josep , Benabarre Antoni
Published: April 2004

We report on a patient who presented with a progressive supranuclear palsy (PSP) syndrome while receiving clebopride (CLB), a prokinetic drug with central antidopaminergic properties. The clinical and neurophysiological signs progressively disappeared after CLB withdrawal. To our knowledge, this is the first published PSP‐like syndrome attributable to an antidopaminergic drug.© 2003 Movement Disorder Society
Solution structure of the palladium(II) complex of 1,4,7,10‐tetrathiacyclododec‐2‐ene‐2,3‐dicarbonitrile
Authors: Holzberger Anja , Kleinpeter Erich
Published: April 2004

The solution structure of the Pd(II) complex of mn‐12‐S4 was studied in detail by NMR spectroscopy. The stoichiometry of the complex was determined by 1H NMR titration experiments. 3J(H,H) coupling constants were extracted from the 2D J‐resolved NMR spectrum of the complex providing information concerning the S—C—C—S torsional angles. Further conclusions about the conformation of [Pd(mn‐12‐S4)](BF4)2 were drawn from experimental NOEs. The results of the NMR study were corroborated by molecular modelling. Copyright © 2004 John Wiley & Sons, Ltd.
Use of herbal drugs in pregnancy: a survey among 400 Norwegian women
Authors: Nordeng Hedvig , Havnen Gro C.
Published: April 2004

Purpose

To investigate the use of herbal drugs by pregnant women.

Methods

We interviewed 400 postpartum women at Ullevål University Hospital in Oslo, Norway about the use of herbal drugs, within 3 days after giving birth by using a structured questionnaire in the period from February to June 2001.

Results

We found that 36% of the pregnant women had used herbal drugs during pregnancy with an average of 1.7 products per woman. The proportion of women using herbal drugs increased throughout the first, second and third pregnancy trimester. The most commonly used herbs were echinacea, iron‐rich herbs, ginger, chamomile and cranberry. Among the women having used herbal drugs in pregnancy, 39% had used herbal drugs that were considered possibly harmful or herbs where information about safety in pregnancy was missing. Herbal galactagogues had been used by 43% of the women who had breastfed a prior child during their breast‐feeding period. Use of herbal drugs in pregnancy had most commonly been recommended by family or friends.

Conclusion

The widespread use of herbal drugs during pregnancy indicates an increased need for documentation about the safety of herbal drugs in pregnancy. To meet the needs of pregnant women, it is necessary for health care personnel to have knowledge about herbal drugs during pregnancy. Copyright © 2004 John Wiley & Sons, Ltd.
A retrospective survey on the safety of Replenate®, a high‐purity factor VIII concentrate
Authors: Gascoigne E. W. , Dash C. H. , Harman C. , Wilmot D.
Published: April 2004

Purpose

To assess the safety of a highly purified, plasma‐derived factor VIII concentrate (Replenate®) in routine clinical use.

Methods

Following guidelines entitled safety assessment of marketed medicines (SAMM), safety data were collected in the UK on 194 patients who received an estimated 47.6 million IU of Replenate®. This population included 47 patients undergoing 53 surgical operations or dental extractions.

Results

The study detected four cases of new factor VIII inhibitor development and twelve other adverse events, five that were unrelated to the product, five whose causality was unknown, one that was possibly product‐related and one case due to possible lack of efficacy. Only one of these cases had been notified to the manufacturer through conventional spontaneous reporting procedures. Three patients were switched from Replenate® as a result of an adverse event (one case of infusion site irritation and two cases of a rise in titre of an existing inhibitor), but no unexpected adverse reactions were noted and there were no reports of virus transmission. The median factor VIII recovery value was 2.17 IU/dl per IU/kg, but recovery was shown to be dependent on several variables, namely inhibitor status, treatment centre and the patient's body weight. The median factor VIII recovery in inhibitor‐free patients was 2.28 IU/dl per IU/kg (range: 1.20–6.62).

Conclusions

The study confirms that Replenate® is well tolerated by the majority of patients in routine clinical practice. © Crown copyright 2004. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.
Catalytic and inhibitory effects of β‐cyclodextrin on the hydrolysis of benzoic anhydride
Authors: Brandão Tiago A. S. , Magro Jacir Dal , Chiaradia Louise D. , Nascimento Maria da Graça , Nome Faruk , Tato Jose V. , Yunes Rosendo A.
Published: April 2004

The hydrolysis of benzoic anhydride (Bz2O) in the presence of β‐cyclodextrin (β‐CD) was studied in aqueous solution as a function of pH, temperature and ionic strength, at 25°C. The experimental rate constant versus pH profiles show that, in the region of spontaneous water reaction (pH 3.0–6.5), β‐CD inhibits the reaction and the isotope effect $(k_{{\rm H_2 O}} / k_{{\rm D_2 O}} = 4.7)$ indicates that the rate determining step of the reaction corresponds to the water‐catalyzed nucleophilic attack of water on the carbonyl group of Bz2O. Conversely, whereas inhibition is observed at pH 6.0, catalysis of the hydroxide ion reaction is observed at pH 8.0 and it is found that the activation entropy is responsible for the catalytic phenomena in the basic hydrolysis of benzoic anhydride. Copyright © 2004 John Wiley & Sons, Ltd.
A large gem‐dimethyl effect in the cyclization of ω‐phenylhydantoic acids: computational modeling of the gem‐dimethyl effect on the acid‐ or base‐catalyzed cyclization of hydantoic acids and esters
Authors: Ivanov P. M. , Pojarlieff I. G. , Blagoeva I. B. , Jaime C. , Angelova V. T. , Koedjikov A. H.
Published: April 2004

The rates of the cyclization of methyl‐substituted 5‐phenylhydantoic acids were measured in acid solutions. A particularly strong gem‐dimethyl effect (GDME) was observed with the N‐methyl compounds amounting to an acceleration of six powers of ten for the 2,2,3‐trimethyl derivative. The variations in the free energies of activation for the cyclization of hydantoic acids and esters were modeled by the strain energies of the tetrahedral intermediates and of the reactants calculated by the MM3 force field. The neutral tetrahedral intermediate T0 was used for reaction series involving acid catalysis and the negatively charged intermediate T− for base catalysis. Very good agreement with the experimental GDME was obtained for the acid‐catalyzed cyclizations of the complete series of the N‐methyl‐substituted substrates, showing that the accelerations result from a greater strain increase in the reactants. The results with T− are closely parallel, indicating that the loss of GDME observed under base catalysis with 2,2,3‐trimethylhydantoate esters is not due to intramolecular strain in T−. A linear correlation (slope 1.22, r = 0.934) is obtained for a plot of the free energy variations against strain energies for the reaction series of 5‐phenylhydantoic acids when the data for the strongly deviating parent acid is excluded. Excellent LFERs are obtained between the reaction series of esters and acids. The observed large rate enhancements induced by N‐substituents explain the switches to cyclization routes in synthetic reactions. Copyright © 2004 John Wiley & Sons, Ltd.
Examination of the lignin content in a softwood and a hardwood decayed by a brown‐rot fungus with the acetyl bromide method and Fourier transform infrared spectroscopy
Authors: Pandey K. K. , Pitman A. J.
Published: April 2004

Fourier transform infrared (FTIR) spectroscopy was used to examine lignin modification in Pinus sylvestris L. (Scots pine) and Fagus sylvatica L. (beech) decayed to different weight losses by the brown‐rot fungus Coniophora puteana. Samples of different weight losses, ranging from 3 to 64% for beech and 15 to 62% for pine, were analyzed along with undecayed controls. The ratios of the heights of the lignin/carbohydrate FTIR peaks were determined, and the lignin contents of the blocks were measured with the acetyl bromide method. The ratios of the reference peaks for lignin against polysaccharide (i.e., cellulose and hemicellulose) peaks were compared with the lignin content of the wood determined by the acetyl bromide method. A good correlation was obtained for ratios of some of the lignin/carbohydrate reference peaks (lignin peaks at 1505 and 1330 cm−1 for beech and at 1511 and 1225 cm−1 for pine against polysaccharide peaks at 1734, 1375, and 1158 cm−1) against the lignin content for both wood species decayed to different levels. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 2340–2346, 2004
Modeling structurally variable regions in homologous proteins with rosetta
Authors: Rohl Carol A. , Strauss Charlie E.M. , Chivian Dylan , Baker David
Published: April 2004

A major limitation of current comparative modeling methods is the accuracy with which regions that are structurally divergent from homologues of known structure can be modeled. Because structural differences between homologous proteins are responsible for variations in protein function and specificity, the ability to model these differences has important functional consequences. Although existing methods can provide reasonably accurate models of short loop regions, modeling longer structurally divergent regions is an unsolved problem. Here we describe a method based on the de novo structure prediction algorithm, Rosetta, for predicting conformations of structurally divergent regions in comparative models. Initial conformations for short segments are selected from the protein structure database, whereas longer segments are built up by using three‐ and nine‐residue fragments drawn from the database and combined by using the Rosetta algorithm. A gap closure term in the potential in combination with modified Newton's method for gradient descent minimization is used to ensure continuity of the peptide backbone. Conformations of variable regions are refined in the context of a fixed template structure using Monte Carlo minimization together with rapid repacking of side‐chains to iteratively optimize backbone torsion angles and side‐chain rotamers. For short loops, mean accuracies of 0.69, 1.45, and 3.62 Å are obtained for 4, 8, and 12 residue loops, respectively. In addition, the method can provide reasonable models of conformations of longer protein segments: predicted conformations of 3Å root‐mean‐square deviation or better were obtained for 5 of 10 examples of segments ranging from 13 to 34 residues. In combination with a sequence alignment algorithm, this method generates complete, ungapped models of protein structures, including regions both similar to and divergent from a homologous structure. This combined method was used to make predictions for 28 protein domains in the Critical Assessment of Protein Structure 4 (CASP 4) and 59 domains in CASP 5, where the method ranked highly among comparative modeling and fold recognition methods. Model accuracy in these blind predictions is dominated by alignment quality, but in the context of accurate alignments, long protein segments can be accurately modeled. Notably, the method correctly predicted the local structure of a 39‐residue insertion into a TIM barrel in CASP 5 target T0186. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Single‐body residue‐level knowledge‐based energy score combined with sequence‐profile and secondary structure information for fold recognition
Authors: Zhou Hongyi , Zhou Yaoqi
Published: April 2004

An elaborate knowledge‐based energy function is designed for fold recognition. It is a residue‐level single‐body potential so that highly efficient dynamic programming method can be used for alignment optimization. It contains a backbone torsion term, a buried surface term, and a contact‐energy term. The energy score combined with sequence profile and secondary structure information leads to an algorithm called SPARKS (Sequence, secondary structure Profiles and Residue‐level Knowledge‐based energy Score) for fold recognition. Compared with the popular PSI‐BLAST, SPARKS is 21% more accurate in sequence‐sequence alignment in ProSup benchmark and 10%, 25%, and 20% more sensitive in detecting the family, superfamily, fold similarities in the Lindahl benchmark, respectively. Moreover, it is one of the best methods for sensitivity (the number of correctly recognized proteins), alignment accuracy (based on the MaxSub score), and specificity (the average number of correctly recognized proteins whose scores are higher than the first false positives) in LiveBench 7 among more than twenty servers of non‐consensus methods. The simple algorithm used in SPARKS has the potential for further improvement. This highly efficient method can be used for fold recognition on genomic scales. A web server is established for academic users on http://theory.med.buffalo.edu. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Ultrahigh resolution drug design I: Details of interactions in human aldose reductase–inhibitor complex at 0.66 Å
Authors: Howard E.I. , Sanishvili R. , Cachau R.E. , Mitschler A. , Chevrier B. , Barth P. , Lamour V. , Van Zandt M. , Sibley E. , Bon C. , Moras D. , Schneider T.R. , Joachimiak A. , Podjarny A.
Published: April 2004

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X‐ray diffraction data were collected up to 0.62 Å resolution and treated up to 0.66 Å resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 Å). The model was very well ordered overall (CA atoms' mean B factor is 5.5 Å2). The model and the electron‐density maps revealed fine features, such as H‐atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B ∼3 Å2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments. Proteins 2004. © 2004 Wiley‐Liss, Inc.
A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV‐1 protease
Authors: Vega Sonia , Kang Lin‐Woo , Velazquez‐Campoy Adrian , Kiso Yoshiaki , Amzel L. Mario , Freire Ernesto
Published: April 2004

The efficacy of HIV‐1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI‐764, a second‐generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI‐577 differs from KNI‐764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV‐1 protease have been determined by X‐ray crystallography at 2.0 Å resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI‐764 to adapt to the mutated binding site cavity more readily than KNI‐577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug‐resistant mutant protease; however KNI‐764 gains binding entropy while KNI‐577 loses binding entropy. The gain in binding entropy by KNI‐764 accounts for its low susceptibility to the drug‐resistant mutation. The heat capacity change associated with binding becomes more negative when KNI‐764 binds to the mutant protease, consistent with increased desolvation. With KNI‐577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI‐764 when it is bound to the drug‐resistant mutant. The opposite is observed for KNI‐577. Consistent with these observations, it appears that KNI‐764 is able to gain binding entropy by a two‐fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease. Proteins 2004. © 2004 Wiley‐Liss, Inc.
The emergence of catalytic and structural diversity within the beta‐clip fold
Authors: Iyer Lakshminarayan M. , Aravind L.
Published: April 2004

The β‐clip fold includes a diverse group of protein domains that are unified by the presence of two characteristic waist‐like constrictions, which bound a central extended region. Members of this fold include enzymes like deoxyuridine triphosphatase and the SET methylase, carbohydrate‐binding domains like the fish antifreeze proteins/Sialate synthase C‐terminal domains, and functionally enigmatic accessory subunits of urease and molybdopterin biosynthesis protein MoeA. In this study, we reconstruct the evolutionary history of this fold using sensitive sequence and structure comparisons methods. Using sequence profile searches, we identified novel versions of the β‐clip fold in the bacterial flagellar chaperone FlgA and the related pilus protein CpaB, the StrU‐like dehydrogenases, and the UxaA/GarD‐like hexuronate dehydratases (SAF superfamily). We present evidence that these versions of the β‐clip domain, like the related type III anti‐freeze proteins and C‐terminal domains of sialic acid synthases, are involved in interactions with carbohydrates. We propose that the FlgA and CpaB‐like proteins mediate the assembly of bacterial flagella and Flp pili by means of their interactions with the carbohydrate moieties of peptidoglycan. The N‐terminal β‐clip domain of the hexuronate dehydratases appears to have evolved a novel metal‐binding site, while their C‐terminal domain is likely to adopt a metal‐binding TIM barrel‐like fold. Using structural comparisons, we show that the β‐clip fold can be further classified into two major groups, one that includes the SAF, SET, dUTPase superfamilies, and the other that includes the phage lambda head decoration protein, the β subunit of urease and the C‐terminal domain of the molybdenum cofactor biosynthesis protein MoeA. Structural comparisons also suggest the β‐clip fold was assembled through the duplication of a three‐stranded unit. Though the three‐stranded units are likely to have had a common origin, we present evidence that complete β‐clip domains were assembled through such duplications, independently on multiple occasions. There is also evidence for circular permutation of the basic three‐stranded unit on different occasions in the evolution of the β‐clip unit. We also describe how assembly of this fold from a basic three‐stranded unit has been utilized to accommodate a variety of activities in its different versions. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Protein–water interactions in ribonuclease A and angiogenin: A molecular dynamics study
Authors: Sanjeev B. S. , Vishveshwara S.
Published: April 2004

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein–water interactions have been carried out on two members of the ribonuclease A superfamily—RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein–water networks are investigated. The locations of such networks are identified for both proteins. Proteins 2004. © 2004 Wiley‐Liss, Inc.
The molecular origin of the thiamin diphosphate‐induced spectral bands of ThDP‐dependent enzymes
Authors: Kovina Marina V. , de Kok Aart , Sevostyanova Irina A. , Khailova Ludmila S. , Belkina Natalya V. , Kochetov German A.
Published: April 2004

New and previously published data on a variety of ThDP‐dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP‐induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non‐enzymatic models with pyrimidine‐like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short‐ and long‐wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1′ atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1′ and the 4′‐nitrogen. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Alternative alignments from comparison of protein structures
Authors: Shih Edward S.C. , Hwang Ming‐Jing
Published: April 2004

Comparison of two protein structures often results in not only a global alignment but also a number of distinct local alignments; the latter, referred to as alternative alignments, are however usually ignored in existing protein structure comparison analyses. Here, we used a novel method of protein structure comparison to extensively identify and characterize the alternative alignments obtained for structure pairs of a fold classification database. We showed that all alternative alignments can be classified into one of just a few types, and with which illustrated the potential of using alternative alignments to identify recurring protein substructures, including the internal structural repeats of a protein. Furthermore, we showed that among the alternative alignments obtained, permuted alignments, which included both circular and scrambled permutations, are as prevalent as topological alignments. These results demonstrated that the so far largely unattended alternative alignments of protein structures have implications and applications for research of protein classification and evolution. Proteins 2004. © 2004 Wiley‐Liss, Inc.
NrdH‐redoxin of Corynebacterium ammoniagenes forms a domain‐swapped dimer
Authors: Stehr Matthias , Lindqvist Ylva
Published: April 2004

NrdH‐redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin‐like amino acid sequence but a thioredoxin‐like activity profile. Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 Å resolution, determined by molecular replacement using E. coli NrdH as model. The structure is the first example of a domain‐swapped dimer from the thioredoxin family. The domain‐swapped structure is formed by an inter‐chain two‐stranded anti‐parallel β‐sheet and is stabilized by electrostatic interactions at the dimer interface. Size exclusion chromatography, and MALDI‐ESI experiments revealed however, that the protein exists as a monomer in solution. Similar to E. coli NrdH‐redoxin and thioredoxin, C. ammoniagenes NrdH‐redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase. However, the loop between α2 and β3, which is complementary to a crevice in the reductase in the thioredoxin–thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C. ammoniagenes NrdH‐redoxin. C. ammoniagenes NrdH‐redoxin has the highly conserved sequence motif W61‐S‐G‐F‐R‐P‐[DE]67 which is unique to the NrdH‐redoxins and which determines the orientation of helix α3. An extended hydrogen‐bond network, similar to that in E. coli NrdH‐redoxin, determines the conformation of the loop formed by the conserved motif. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Density functional theory study on isomerization reactions of pyridazine derivatives bearing N,N‐dialkylaminoethyl group
Authors: Zhou Ziyan , Pan Xiumei , Wu Xue , Su Zhongmin , Kan Yuhe , Xie Yuzhong
Published: April 2004

The isomerization reactions of four pyridazine derivatives, i.e., 6‐chloro‐3‐hydroxy‐pyridazine, 1,2‐dihydrogen‐1‐methyl‐3,6‐pyridazindione, 6‐chloro‐3‐diethylamino‐ethoxy‐pyridazine, and 1‐methyl‐2‐diethylaminoethyl‐3,6(2H)‐pyridazindione, have been studied using density functional theory (DFT) B3LYP method at the 6‐311+G** basis set. The geometric structures of reactants, transition states and products have been optimized. The transition states have been explored and proved by vibration analysis as well as intrinsic reaction coordination (IRC) analysis. The activation energies of four reactions were calculated, and the corresponding isomerization mechanisms were also elucidated in theory. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004
Comparison of HF, HF + MP2, LDA, BLYP, and B3LYP band structures of the homopolypeptides
Authors: Ladik J. , Bogar F. , Penke B.
Published: April 2004

Ab initio HF, HF + MP2, LDA DFT, BLYP DFT, and B3LYP DFT calculations are compared in the case of 19 homopolypeptides in their β pleated sheet conformation. The results show that the B3LYP method provides good results for the fundamental gaps, as compared with the values estimated on the basis of available UV spectra and intermediate exciton calculations for PolyGly and PolyAla. The HF method gives the best agreement, using Koopman's theorem for the ionization potential, taking the calculated VBmax values in the HF case if one compares them with the experimental ionization potentials of the 19 amino acids measured by mass spectroscopy. Finally, how these methods might be improved to determine the most stable conformations of the homopolypeptides is outlined. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004
A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection
Authors: Lee Sang Hee , Jung Byung Hwa , Kim Sun Yeou , Chung Bong Chul
Published: April 2004

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high‐performance liquid chromatography (HPLC) with single‐parameter detectors like electron‐capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI‐MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI‐MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive‐ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2–400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03–14.92% (inter‐day) and 0.54–14.39% (intra‐day), respectively. Copyright © 2004 John Wiley & Sons, Ltd.
A mass spectrometric and molecular orbital study of H2O loss from protonated tryptophan and oxidized tryptophan derivatives
Authors: Lioe Hadi , O'Hair Richard A. J. , Reid Gavin E.
Published: April 2004

Protonated N‐acetyltryptophan, oxindolylalanine (a mono‐oxidized derivative of tryptophan), and N‐acetyloxindolylalanine, as well as several di‐ and tripeptide derivatives containing oxindolylalanine, undergo a range of fragmentation reactions in the gas phase, including the loss of water. In order to elucidate the sites of water loss within these ions, and to determine the mechanisms associated with these processes, we have conducted a series of experiments employing multistage tandem mass spectrometry (MS/MS and MS3) in a quadrupole ion trap mass spectrometer, regiospecific structural labeling, and independent solution‐phase syntheses of proposed product ion structures, coupled with the use of molecular orbital calculations at the B3LYP/6‐31G* level of theory. We demonstrate that the loss of H2O from the amide carbonyl group of protonated N‐acetyltryptophan O‐methyl ester occurs via a ‘side‐chain–backbone' neighboring group reaction to yield a protonated carboline derivative. In contrast, the loss of water from the O‐methyl ester of protonated oxindolylalanine results in the formation of a tricyclic structure by ‘backbone–side‐chain’ nucleophilic attack from the amino nitrogen to the C2 position of the indole ring. The O‐methyl ester of protonated N‐acetyloxindolylalanine was found to dissociate via the loss of water from both possible sites, i.e. from the side‐chain indolyl oxygen and the backbone amide carbonyl group. An estimate of the relative preference for water loss from each site was obtained from the abundances of product ions formed from MS3 analysis of regiospecifically labeled derivatives of N‐acetyloxindolylalanine, and from the results of molecular orbital calculations. These studies indicate the absence of a characteristic ‘signature’ ion or neutral loss for peptides containing oxindolylalanine residues under low‐energy ion trap CID conditions. Copyright © 2004 John Wiley & Sons, Ltd.