bluuebunny/crossref_metadata_2025 huggingface dataset:
CO‐Free Hydrogen Production by Ethanol Steam Reforming in a Pd–Ag Membrane Reactor
Authors: Basile A. , Gallucci F. , Iulianelli A. , Tosti S.
Published: January 2008

In this work, the ethanol steam reforming (ESR) reaction has been studied by using a dense Pd–Ag membrane reactor (MR) by varying the water/ethanol molar ratio between 3:1 and 9:1 in a temperature range of 300–400 °C and at 1.3 bar as reaction pressure. The MR was packed with a commercial Ru‐based catalyst and a constant sweep gas flow rate in counter current mode was used. The influence of the temperature and the feed molar ratio on different parameters such as the ethanol conversion, the hydrogen production, the hydrogen yield and the CO‐free hydrogen recovery has been evaluated.
Two‐Phase 1D+1D Model of a DMFC: Development and Validation on Extensive Operating Conditions Range
Authors: Casalegno A. , Marchesi R. , Parenti D.
Published: January 2008

A two‐phase 1D+1D model of a direct methanol fuel cell (DMFC) is developed, considering overall mass balance, methanol transport in gas phase through anode diffusion layer, methanol and water crossover. The model is quantitatively validated on an extensive range of operating conditions, 24 polarisation curves. The model accurately reproduces DMFC performance in the validation range and, outside this, it is able to predict values under feasible operating conditions. Finally, the estimations of methanol crossover flux are qualitatively and quantitatively similar to experimental measures and the main local quantities' trends are coherent with results obtained with more complex models.
Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation
Authors: Barlič Ariana , Drobnič Matej , Maličev Elvira , Kregar‐Velikonja Nevenka
Published: January 2008

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0–10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real‐time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three‐dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension–periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847–853, 2008
Embedding vector‐valued Besov spaces into spaces of γ ‐radonifying operators
Authors: Kalton Nigel , van Neerven Jan , Mark Mark , Weis Lutz
Published: January 2008

It is shown that a Banach space E has type p if and only for some (all) d ≥ 1 the Besov space B(1/p – 1/2)d p,p (ℝd ; E) embeds into the space γ (L2(ℝd ), E) of γ ‐radonifying operators L2(ℝd ) → E. A similar result characterizing cotype q is obtained. These results may be viewed as E ‐valued extensions of the classical Sobolev embedding theorems. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Abnormal metabolic networks in atypical parkinsonism
Authors: Eckert Thomas , Tang Chengke , Ma Yilong , Brown Nathaniel , Lin Tanya , Frucht Steven , Feigin Andrew , Eidelberg David
Published: January 2008

Spatial covariance analysis has been used with 18F‐fluorodeoxyglucose (FDG) PET to detect and quantify specific metabolic patterns associated with Parkinson's disease (PD). However, PD‐related patterns cannot necessarily serve as biomarkers of the processes that underlie the atypical parkinsonian syndromes. In this FDG PET study, we used strictly defined statistical criteria to identify disease‐related metabolic patterns in the imaging data from patients with multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), the two most common of these atypical conditions. We found that MSA and PSP were each associated with a specific, highly stable metabolic brain network (P < 0.0001, bootstrap estimation). The MSA‐related pattern was characterized by decreased metabolism in the putamen and cerebellum. The PSP‐related pattern was characterized by metabolic decreases in the brainstem and medial frontal cortex. For both conditions, pattern expression was significantly elevated in patients relative to age‐matched healthy control subjects (P < 0.001). For each condition, we validated the associated disease‐related metabolic pattern by computing its expression on an individual scan basis in two independent patient cohorts, and in one subsequent healthy volunteer cohort. We found that for both MSA and PSP, prospective assessments of pattern expression accurately discriminated patients from controls (P < 0.001). These findings suggest that the major atypical parkinsonian syndromes are associated with distinct patterns of abnormal regional metabolic activity. These disease‐related networks can potentially be used in conjunction with functional brain imaging as quantifiable biomarkers for the assessment of these pathological conditions. © 2008 Movement Disorder Society
Inconsistencies between reported test statistics and p‐values in two psychiatry journals
Authors: Berle David , Starcevic Vladan
Published: January 2008

A recent survey of the British Medical Journal (BMJ) and Nature revealed that inconsistencies in reported statistics were common. We sought to replicate that survey in the psychiatry literature. We checked the consistency of reported t‐test, F‐test and χ2‐test values with their corresponding p‐values in the 2005 issues of the Australian and New Zealand Journal of Psychiatry (ANZJP) and compared this with the issues of the ANZJP from 2000, and with a similar journal, Acta Psychiatrica Scandinavica (APS). A reported p‐value was ‘inconsistent’ if it differed (at its reported number of decimal places) from our calculated p‐values (using three different software packages), which we based on the reported test statistic and degrees of freedom. Of the 546 results that we checked, 78 (14.3%) of the p‐values were inconsistent with the corresponding degrees of freedom and test statistic. Similar rates of inconsistency were found in APS and ANZJP, and when comparing the ANZJP between 2000 and 2005. The percentages of articles with at least one inconsistency were 8.5% for ANZJP 2005, 9.9% for ANZJP 2000 and 12.1% for APS. We conclude that inconsistencies in p‐values are common and may reflect errors of analysis and rounding, typographic errors or typesetting errors. Suggestions for reducing the occurrence of such inconsistencies are provided. Copyright © 2008 John Wiley & Sons, Ltd.
Using decision theory to derive optimal cut‐off scores of screening instruments: an illustration explicating costs and benefits of mental health screening
Authors: Smits Niels , Smit Filip , Cuijpers Pim , De Graaf Ron
Published: January 2008

This paper shows how decision theory can be used to determine optimal cut‐off scores on mental health screeners. The procedure uses (a) the costs and benefits of correct and erroneous decisions, and (b) the rates of correct and erroneous decisions as a function of the cut‐off score. Using this information, for each cut‐off point expected costs are calculated. The cut‐off point with the lowest expected costs is the optimal cut‐off score. An illustration is given in which the General Health Questionnaire is employed as a major depression screener. Optimal cut‐off points are determined for four different contexts: patients, health service providers, society, and mental health researchers. As in these four situations different costs are encountered, different optimal cut‐off points were found. Copyright © 2008 John Wiley & Sons, Ltd.
Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22) with high rates of unbalanced sperm and embryos: a case report
Authors: Wiland Ewa , Hobel Calvin J. , Hill David , Kurpisz Maciej
Published: January 2008

Background

Regarding the literature on the results of preimplantation genetic diagnosis (PGD) in reciprocal chromosomal translocation carriers seems to prevail a view that this method reduces the frequency of miscarriages, and the pregnancy rate is directly proportional to the number of normal spermatozoa. Therefore, we compared the results of sperm karyotype analysis of a carrier of familial t(2;7)(p11.2;q22) with PGD results. The carrier was ascertained as his wife had had two miscarriages.

Methods

Empirical data from a pedigree of t(2;7)(p11.2;q22) carrier was collected. A tri‐color fluorescence in situ hybridization method (FISH) was used to show the meiotic segregation pattern in sperm of the proband. PGD of blastomeres from a single ICSI cycle and standard prenatal diagnosis procedures to confirm the PGD results was performed.

Results

Meiotic segregation pattern showed only 34.2% of normal/balanced spermatozoa. The high rate (42%) of miscarriages was observed in this family, which could be explained by chromosomal unbalanced karyotypes as a product of fertilization by unbalanced spermatozoa found with a frequency of approximately 66%. The lack of unbalanced progeny at birth suggests a natural selection of unbalanced fetuses. The 37.5% of normal/balanced embryos received after a single ICSI cycle and PGD was similar to the percentage of normal/balanced spermatozoa (34.2%). After 38 weeks a healthy girl with normal karyotype was born.

Conclusion

The presented study is an optimistic message for translocation carriers showing that even in case with more than 60% of genetically unbalanced sperm there is a reasonable chance for reproductive success. Copyright © 2008 John Wiley & Sons, Ltd.
Prenatal diagnosis of Roberts syndrome and detection of an ESCO2 frameshift mutation in a Pakistani family
Authors: Schulz Solveig , Gerloff Claudia , Ledig Susanne , Langer Dorothea , Volleth Mariannne , Shirneshan Katayoon , Wieacker Peter
Published: January 2008

Objectives

We report two siblings with Roberts syndrome (RBS), and an attempt to delineate the underlying molecular mechanism leading to familial recurrence.

Methods

Cytogenetic studies and direct sequencing of the ESCO2 gene were carried out in the second affected fetus and the parents. Fetal DNA was obtained from amniocytes after amniocentesis. Parental DNA was obtained from peripheral blood samples.

Results

Cytogenetic analysis of amniocytes revealed a normal male karyotype in 20 analyzed metaphases and chromosomal aneuploidies in 10 metaphases. All metaphases displayed premature separation of centromeres and puffing of heterochromatic regions near the centromere. A homozygous mutation leading to a frameshift in ESCO2 was identified in the fetal DNA sample. Both parents are heterozygous carriers of the same mutation.

Conclusion

The present case demonstrates the prenatal diagnosis of RBS associated with a frameshift mutation in ESCO2. Copyright © 2008 John Wiley & Sons, Ltd.
Sonographic assessment of the extra‐abdominal fetal small bowel in gastroschisis: a retrospective longitudinal study in relation to prenatal complications
Authors: Heinig J. , Müller V. , Schmitz R. , Lohse K. , Klockenbusch W. , Steinhard J.
Published: January 2008

Objective

An explorative retrospective study following a case‐series of fetuses with isolated gastroschisis, to evaluate if small‐bowel dilatation may be indicative for emerging obstetric complications. The secondary aim was to establish preliminary normative curves for the external diameter and wall thickness of eventerated fetal small bowel in gastroschisis during the second and third trimester of pregnancy.

Methods and Materials

Fourteen fetuses with isolated gastroschisis were followed at a single center. Repeated ultrasound examinations for fetal surveillance with measurement of fetal small‐bowel diameter and wall thickness over the course of pregnancy until delivery were performed.

Results

Longitudinal data analysis showed significantly increasing bowel diameter and wall thickness of eventerated small bowel with advancing gestation. Dilatation of small bowel more than 25 mm in the third trimester of pregnancy was associated with an increased risk of short‐term prenatal complications as fetal distress or intrauterine fetal death (PPV 100%; 95% CI: 29.2–100%, NPV 100%; 95% CI: 71.5–100%).

Conclusions

Dilatation of the extra‐abdominal fetal small bowel in the third trimester may allow identifying fetuses with increased risk of fetal distress requiring closer monitoring of fetal well‐being or delivery in a short interval to prevent impending fetal death. Copyright © 2008 John Wiley & Sons, Ltd.
An insight into the general relationship between the three dimensional structures of enzymes and their electronic wave functions: Implication for the prediction of functional sites of enzymes
Authors: Fukushima K. , Wada M. , Sakurai M.
Published: January 2008

In this study, we explored the general relationship between the three‐dimensional (3D) structures of enzymes and their electronic wave functions. Furthermore, we developed a method for the prediction of their functionally important sites. For this purpose, we first performed linear‐scaling molecular orbital calculations for 112 nonredundant, non‐homologous enzymes with known structure and function. In consequence, we showed that the canonical molecular orbitals (MOs) of the enzymes could be classified into three groups according to the degree of electron delocalization: highly localized orbitals (Group A), highly delocalized orbitals whose electrons are distributed over almost the whole molecule (Group B), and moderately delocalized orbitals (Group C). The MOs belonging to Group A are located near the HOMO‐LUMO band gap, and thereby include the frontier orbitals of a given enzyme. We inferred that the MOs of Group B play a role in stabilizing the 3D structure of the enzyme, while those of Group C contribute to constructing the covalent bond framework of the enzyme. Next, we investigated whether the frontier orbitals of enzymes could be used for identifying their potential functional sites. As a result, we found that the frontier orbitals of the 112 enzymes have a high propensity to be colocalized with the known functional sites, especially when the enzymes are hydrated. Such a propensity is shown to be remarkable when Glu or Asp is a functional site residue. On the basis of these results, we finally propose a protocol for the prediction of functional sites of enzymes. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Stacking efficiency and flexibility analysis of aromatic amino acids in cap‐binding proteins
Authors: Worch Remigiusz , Stolarski Ryszard
Published: January 2008

Recognition of the ribonucleic acid 5′ termini (RNA 5′ cap) by a wide class of cap‐binding proteins is largely accomplished by cation‐π stacking that involves the positively charged 7‐methylguanine ring and aromatic amino acid side chains. Quantum calculations of the stacking energy were performed by means of MP2 perturbation method for binary and ternary associates composed of the 7‐methylguanine moiety and tryptophan, tyrosine, or phenylalanine, in their spatial orientations known from the crystalline cap‐protein complexes. The results clearly pointed to an enhancement of the stacking energy due to a net positive charge in the cap guanine moiety and allowed analysis of a role of various amino acids in stabilization of the complexes. Conformational flexibility of the aromatic amino acids taking part in binding ligands to a wide class of RNA‐recognizing proteins, including the cap‐binding proteins, was determined by regional order neural network (RONN) algorithm that provides results close to those of the crystallographic B‐factors analysis. Interestingly, some of the tyrosines that are classified in general as “rigid” showed high flexibility when engaged in binding the cap to nuclear cap‐binding protein complex CBC and to viral methyltransferase VP39. Parallel analyses of the binding energy and flexibility of the protein fragments engaged in the binding leads to understanding differences in molecular mechanisms of the cap recognition by various proteins, CBC compared with the eukaryotic initiation factor eIF4E, and enzymes vs. other protein factors. Proteins 2008. © 2008 Wiley‐Liss, Inc.
A phospho‐sugar binding domain homologous to NagB enzymes regulates the activity of the central glycolytic genes repressor
Authors: Doan Thierry , Martin Laetitia , Zorrilla Silvia , Chaix Denis , Aymerich Stéphane , Labesse Gilles , Declerck Nathalie
Published: January 2008

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C‐terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged‐helix DNA‐binding motif followed by a C‐terminal domain presenting weak but significant homology with glucosamine‐6‐phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C‐terminal domain would bind preferentially bi‐phosphorylated compounds, in agreement with previous studies that proposed fructuose‐1,6‐biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono‐ and di‐phospho‐fructosides. Based on these results, we propose that the activity of the CggR‐like repressors is controlled by a phospho‐sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Structural basis for the cold adaptation of psychrophilic M37 lipase fromPhotobacterium lipolyticum
Authors: Jung Suk‐Kyeong , Jeong Dae Gwin , Lee Mi Sook , Lee Jung‐Kee , Kim Hyung‐Kwoun , Ryu Seong Eon , Park Byoung Chul , Kim Jae Hoon , Kim Seung Jun
Published: January 2008

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 Å resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures. Proteins 2008. © 2008 Wiley‐Liss, Inc.
The solution structure of the outer membrane lipoprotein OmlA from Xanthomonas axonopodis pv. citri reveals a protein fold implicated in protein–protein interaction
Authors: Vanini Marina Marques Teixeira , Spisni Alberto , Sforça Maurício Luis , Pertinhez Thelma Aguiar , Benedetti Celso Eduardo
Published: January 2008

The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant‐pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N‐ and C‐ termini and a structurally well defined core composed of three β‐strands and two small α‐helices, which pack against each other forming a two‐layer alpha/beta scaffold. This protein fold resembles the domains of the β‐lactamase inhibitory protein BLIP, involved in protein–protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein–protein interactions required during X. citri infection. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Development of new laccases by directed evolution: Functional and computational analyses
Authors: Festa Giovanna , Autore Flavia , Fraternali Franca , Giardina Paola , Sannia Giovanni
Published: January 2008

Laccases are blue multicopper oxidases that couple the four‐electron reduction of oxygen with the oxidation of a broad range of aromatic substrates. These fungal enzymes can be used for many applications such as bleaching, organic synthesis, bioremediation, and in laundry detergents. Laccases from Pleurotus ostreatus have been successfully heterologously expressed in yeasts. The availability of established recombinant expression systems has allowed the construction of mutated, “better performing” enzymes through molecular evolution techniques. In the present work, random mutagenesis experiments on poxc and poxa1b cDNAs, using error prone PCR (EP‐PCR) have been performed. By screening a library of 1100 clones the mutant 1M9B was selected, it shows a single mutation (L112F) leading to an enzyme more active but less stable with respect to the wild‐type enzyme (POXA1b) in all the analyzed conditions. This mutant has been used as a template for a second round of EP‐PCR. From this second generation library of 1200 clones, three mutants have been selected. Properties of the four mutants, 1M9B screened from the first library, and 1L2B, 1M10B, and 3M7C from the second library, were analyzed. The better performing mutant 3M7C presents, besides L112F, only one substitution (P494T) responsible both for the significantly increased stability and for the exhibited higher activity of this mutant. Molecular dynamics simulations have been performed on three‐dimensional models of POXA1b, 1M9B, and 3M7C, and hypotheses on the structure–function relationships of these proteins have been formulated. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax
Authors: Pauluhn Anuschka , Ahmed Hatim , Lorentzen Esben , Buchinger Sebastian , Schomburg Dietmar , Siebers Bettina , Pohl Ehmke
Published: January 2008

Carbon–carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of CC bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2‐keto‐3‐deoxy‐(6‐phospho)‐gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden–Meyerhof–Parnas pathway and an unusual branched Entner–Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2‐keto‐3‐deoxy‐6‐phosphogluconate (KDPG) and 2‐keto‐3‐deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3‐phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2‐keto‐3‐deoxy‐6‐phosphogalactonate (KDPGal) and 2‐keto‐3‐deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 Å, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 Å. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme apparently also lacks stereo control, suggesting that the enzyme is a trade‐off between required catabolic flexibility needed for the conversion of phosphorylated and nonphosphorylated substrates and required stereo control of cellular/physiological enzymatic reactions. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Molecular dynamics simulations of the active matrix metalloproteinase‐2: Positioning of the N‐terminal fragment and binding of a small peptide substrate
Authors: Díaz Natalia , Suárez Dimas
Published: January 2008

Herein we use different computational methods to study the structure and energetic stability of the catalytic domain of the active MMP‐2 enzyme considering two different orientations of its N‐terminal coil. The first orientation is largely solvent accessible and corresponds to that observed in the 1CK7 crystal structure of the proenzyme. In the second orientation, the N‐terminal coil is packed against the Ω‐loop and the α3‐helix of the MMP‐2 enzyme likewise in the so‐called “superactivated” form of other MMPs. Binding to the MMP‐2 catalytic domain of a short peptide substrate, which mimics the sequence of the α1 chain of collagen type I, is also examined considering again the two configurations of the N‐terminal coil. All these MMP‐2 models are subject to 20 ns molecular dynamics (MD) simulations followed by MM‐PBSA (Molecular Mechanics Poisson‐Boltzmann Surface Area) calculations. The positioning of the N‐terminal coil in the “superactivated” form is found to be energetically favored for the MMP‐2 enzyme. Moreover, this configuration of the N‐terminal moiety can facilitate the binding of peptide substrates. Globally, the results obtained in this study could be relevant for the structural‐based design of specific MMP inhibitors. Proteins 2008. © 2008 Wiley‐Liss, Inc.
The X‐ray structure ofN‐methyltryptophan oxidase reveals the structural determinants of substrate specificity
Authors: Ilari Andrea , Bonamore Alessandra , Franceschini Stefano , Fiorillo Annarita , Boffi Alberto , Colotti Gianni
Published: January 2008

The X‐ray structure of monomeric N‐methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 Å resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N‐methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in kcatand 100 fold decrease in Kmfor sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three‐fold decrease in activity towards N‐methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308‐FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Ordered conformational change in the protein backbone: Prediction of conformationally variable positions from sequence and low‐resolution structural data
Authors: Kuznetsov Igor B.
Published: January 2008

Ordered conformational changes are an important structural property of proteins and are involved in a variety of fundamental biological activities. Large‐scale analyses of the implications of such changes for protein function and dysfunction require efficient methods for automated recognition of conformationally variable residue positions. The goal of this work was to study sequence and low‐resolution structural properties of residue positions that change backbone conformation upon changes in protein environment and the utility of these properties for automated recognition of such conformationally variable positions. This study was performed using a large nonredundant set of experimentally characterized proteins that undergo ordered conformational transitions obtained from the Database of Macromolecular Movements. The results of this study show that ordered changes in backbone conformation are not limited to solvent accessible loop regions. A considerable fraction of conformationally variable positions is observed in helices and strands, and in buried positions. Conformationally variable positions are less conserved in evolution. Local patterns of (a) sequence neighbors, (b) evolutionary conservation, and (c) solvent accessibility can be used to predict conformationally variable positions with balanced sensitivity and specificity, albeit with large variance at the level of individual proteins. However, including a pattern of secondary structure into the prediction scheme results in a highly unbalanced performance when all conformationally variable positions located in regular secondary structure are misclassified. Application of the present methodology to the prion protein (PrP) shows that conformationally variable positions predicted in its ordered C‐terminal domain are located within segments presumed to be involved in refolding of PrP. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Sequence–structure–function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA
Authors: Roovers Martine , Oudjama Yamina , Kaminska Katarzyna H. , Purta Elzbieta , Caillet Joël , Droogmans Louis , Bujnicki Janusz M.
Published: January 2008

MnmC catalyses the last two steps in the biosynthesis of 5‐methylaminomethyl‐2‐thiouridine (mnm5s2U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by theyfcKopen reading frame in theEscherichia coliK12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold‐recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site‐directed and deletion mutagenesis, respectively. We found out that the N‐terminal domain contains residues responsible for binding of the S‐adenosylmethionine cofactor and catalyzing the methylation of nm5s2U to form mnm5s2U, while the C‐terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N‐terminal domain is capable of independent folding, while the isolated C‐terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC. Proteins, 2008. © 2008 Wiley‐Liss, Inc.
On defining the dynamics of hydrophobic patches on protein surfaces
Authors: Lijnzaad Philip , Feenstra K. Anton , Heringa Jaap , Holstege Frank C. P.
Published: January 2008

We present a simple and efficient method called PATCHTRACK, for studying the dynamics of hydrophobic surface patches. It tracks the patches on snapshot structures taken from a Molecular Dynamics simulation. They are connected into so‐called patch runs, which are subsequently clustered into so‐called recurrent patches. The method is applied to simulations of three different proteins. Protein motion causes addition and removal of one or more atoms to a patch, resulting in size fluctuations of around 25%. The fluctuations eventually lead to the break‐up of a patch, and their average life span is therefore remarkably short at around 4 ps. However, some patch runs are much more stable, lasting hundreds of picoseconds. One such case is the largest patch in amicyanin that is known to be biologically relevant. Another case, previously not reported, is found in phospholipase A2, where the functional significance of a large recurrent patch formed by Leu58 and Phe94 seems likely. This patch appears to have been overlooked as it is relatively small in the X‐ray structure, demonstrating the utility of the current method. The most frequently occurring patch size is 40–60 Å2, but sizes of up to 500 Å2 are also observed. There is no clear relation between patch run durations and their average size. However, long‐lasting patch runs tend not to have large fluctuations. The recurrent patches have alternating periods of “liveness” and “dormancy”; around 25% of them is predominantly in the live state. Proteins 2008. © 2008 Wiley‐Liss, Inc.
Four new forms of the contracted Schrödinger equation and their connection with the second‐order hypervirial condition
Authors: Valdemoro C. , Tel L. M. , Pérez‐Romero E. , Alcoba D. R.
Published: January 2008

Through the use of an important property of a fourth‐order correlation matrix previously reported (Valdemoro, C.; Tel, L. M.; Alcoba, D. R.; Perez‐Romero, E.; Casquero, F. J. Int J Quantum Chem 2002, 90, 1555; Alcoba, D. R.; Valdemoro, C. Phys Rev A 2001, 64, 062105) four new equivalent forms of the second‐order contracted Schrödinger equation (2‐CSE) are obtained. The role played by the energy terms involving these correlation matrices is crucial in the solution of these equations. The relations linking the Hermitian (Yasuda, K. Phys Rev A 1999, 59, 4133) and antiHermitian parts (Mazziotti, D. A. Phys Rev Lett 2006, 97, 142002) of the 2‐CSE with these correlation energy terms are analyzed in detail. A discussion of the second‐order hypervirial condition is also given here. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008
Twist‐dependent stacking energy of base‐pair steps in B‐DNA geometry: A density functional theory approach
Authors: Samanta Sudipta , Kabir Mukul , Sanyal Biplab , Bhattacharyya Dhananjay
Published: January 2008

Stacking energy of all the 10 unique DNA base‐pair steps (bp step) are calculated using density functional theory within the ultrasoft pseudopotential plane wave method and local density approximation for the exchange‐correlation functional. We have studied the dependence of stacking energy on twist angle, an aspect found difficult to explain using classical theory. We have found that the twist angle for different bp steps at stacking energy minimum matches extremely well with the values of average twist obtained from B‐DNA crystal structure data. This indicates that the use of a proper quantum chemical method to calculate the π‐π electronic interactions may explain stacking energy without incorporating hydrophobic interaction through solvent or effect of backbone through pseudobond. From the twist angle‐dependent stacking energy profile, we have also generated the probability distributions of twist for all the bp steps and calculated the variance of the distribution. Our calculated variances show similar trend to that of the experimental data for which sufficient numbers of data are available. The TA, AT, and CG doublets show large variances among the 10 possible bp steps, indicating their maximum flexibility. This might be the case of unusual deformation observed at the TATA‐box while binding to TBP protein. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008
Hydrogen bonding characterization of XH2NH2···HNO(X = B, Al, Ga) complexes: A theoretical investigation
Authors: Liu Ying
Published: January 2008

The complexes of XH2NH2···HNO(X = B, Al, Ga) are characterized as head to tail with hydrogen bonding interactions. The structural characteristics can be confirmed by atoms in molecules (AIM) analysis, which also provide comparisons of hydrogen bonds strengths. The calculated interaction energies at G2MP2 level show that stability of complexes decrease as BH2NH2···HNO > AlH2NH2···HNO > GaH2NH2···HNO. On the basis of the vibrational frequencies calculations, there are red‐shifts for ν(X1H) and blue‐shifts for ν(NH) in the complexes on dihydrogen bonding formations (X1H···HN). On hydrogen bonding formations (NH···O), there are red‐shifts for ν(NH) compared to the monomers. Natural bond orbital (NBO) analysis is used to discuss the reasons for the ν(X1H) and ν(NH) stretching vibrational shifts by hyperconjugation, electron density redistribution, and rehybridization. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008
ONIOM investigation of nucleotide selectivity in phosphodiesterases 3 and 4
Authors: Salter E. A. , O'Brien Kerrie A. , Edmunds R. Wesley , Wierzbicki A.
Published: January 2008

The cyclic nucleotide phosphodiesterases (PDEs) are drug‐targeted enzymes that down regulate cyclic nucleotide concentrations in the cell by catalyzing the hydrolysis of the O3′‐phosphorous bond, yielding the noncyclic nucleotides. Selectivity for cAMP versus cGMP (cyclic 3′,5′‐adenosine/‐guanosine monophosphate) as the favored substrate is primarily attributed to the orientation of a conserved glutamine residue which binds to the adenine/guanine ring. We use ONIOM hybrid quantum methods to accurately describe substrate binding within the catalytic sites of the cAMP‐specific PDE4 and the cGMP‐inhibited, dual‐specific PDE3 in order to understand subtle aspects of substrate selectivity. We estimate PDE4's net preference for cAMP binding to be about 16 kcal/mol; the cause of cAMP's known preference resides both in its fixed glutamine orientation (Gln 369 in PDE4D) and in the differential free energy of solvation, which disfavors the binding of cGMP relative to cAMP by about 15 kcal/mol. Also, we discuss the contributing role played by Asn 321, held in place by a partner Asp 167, in the deselection of cGMP by PDE4. PDE3's conserved glutamine (Gln 988 in PDE3B) is free to take on either a cGMP‐favorable or cAMP‐favorable orientation. We find that enthalpies of binding favor cGMP for PDE3, but only by the same amount as free energies of solvation disfavor cGMP binding. Comparison of the PDE3‐cAMP and ‐cGMP complexes and energetics reveals cAMP to be more susceptible to the attack of the hydroxide nucleophile in PDE3. We identify a key threonine residue (Thr 952) as responsible for PDE3's kinetic relative disfavor of cGMP hydrolysis by causing Gln 988 to tilt out of cGMP's purine plane. Our results are consistent with the PDE3's kinetic specificity for cAMP hydrolysis and the known competitive inhibition of PDE3 by cGMP. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008
The design, enactment, and experience of inquiry‐based instruction in undergraduate science education: A case study
Authors: Park Rogers Meredith A. , Abell Sandra K.
Published: January 2008

The purpose of this study was to understand one case of undergraduate inquiry‐based instruction through the words and actions of students and instructors. The data sources included fieldnotes from 16 of 29 classes, two sets of student and instructor interviews (beginning and end of the semester), and a collection of artifacts, such as the laboratory manual, lecture handouts, and the course syllabus. The participants for this study included four faculty instructors and two purposively selected student groups, totaling eight students. We found the instructors' two course goals, (a) teaching students how scientists do science and (b) using an interdisciplinary approach to develop students' content knowledge of the big ideas in science, were consistent with our observations and the students' descriptions of their experience in the course. However, we observed, and the students also noted, an important feature of the course that the instructors did not describe as a course goal was its reliance on the social nature of learning. This telling case demonstrates that inquiry‐based instruction is achievable in undergraduate science education. Implications are discussed for college science instructors interested in inquiry teaching. © 2008 Wiley Periodicals, Inc. Sci Ed 92:591–607, 2008
Analysis of UDP‐galactose 4′‐epimerase mutations associated with the intermediate form of type III galactosaemia
Authors: Chhay J. S. , Vargas C. A. , McCorvie T. J. , Fridovich‐Keil J. L. , Timson D. J.
Published: January 2008

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP‐galactose 4′‐epimerase (GALE). Traditionally, the condition has been divided into two forms—a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose‐sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, kcat) compared with the wild‐type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose‐sensitivity of gal10Δ cells as fully as was the wild‐type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.
Viscero‐somatic reflexes in referred pain areas evoked by capsaicin stimulation of the human gut
Authors: Arendt‐Nielsen Lars , Schipper Klaus‐Peter , Dimcevski Georg , Sumikura Hiroyuki , Krarup Anne Lund , Giamberardino Maria Adele , Drewes Asbjørn Mohr
Published: January 2008

The interaction between visceral pain and the sympathetic nervous system is only sparsely investigated in quantitative human studies. Referred visceral pain can be evoked experimentally by application of substances such as capsaicin (the pungent substance of chilli pepper) to the gut. The aim of the present study was to induce referred visceral pain from the small and large intestine in 32 volunteers via the stomal opening in patients with ileo‐ or colostomy and quantify the viscero‐somatic reflex responses in these referred pain areas by thermography and laser doppler flowmetry.

Capsaicin evoked pain and referred pain areas in all subjects. In the referred pain area, the temperature increased by approximately 0.6°C (P<0.001) and the blood flow by approximately 35AU (P<0.001). Saline was used in a control experiment, and no temperature and blood flow changes were found.

The present quantitative human study of viscero‐somatic reflexes showed dramatic sympathetic responses in the referred pain areas after experimentally induced gut pain.
Ellagic acid suppresses oxidised low-density lipoprotein-induced aortic smooth muscle cell proliferation: studies on the activation of extracellular signal-regulated kinase 1/2 and proliferating cell nuclear antigen expression
Authors: Chang Weng-Cheng , Yu Ya-Mei , Chiang Su-Yin , Tseng Chiung-Yao
Published: January 2008

Proliferation of intimal vascular smooth muscle cells is an important component in the development of atherosclerosis. Ellagic acid is a phenolic compound present in fruits (raspberries, blueberries, strawberries) and walnuts. The present study investigated the effect of ellagic acid on the oxidised LDL (ox-LDL)-induced proliferation of rat aortic smooth muscle cells (RASMC). The study found that ellagic acid significantly inhibited ox-LDL-induced proliferation of RASMC and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2.Furthermore, ellagic acid also blocked the ox-LDL-induced (inducible) cell-cycle progression and down regulation of the expression of proliferating cell nuclear antigen (PCNA) in RASMC. Therefore, ellagic acid reduced the amount of ox-LDL-induced proliferation of RASMC via inactivation of the ERK pathway and suppression of PCNA expression. These results may significantly advance the understanding of the role that antioxidants play in the prevention of atherosclerosis.