bluuebunny/crossref_metadata_2025 huggingface dataset:
Sleeping Beauty transposase modulates cell-cycle progression through interaction with Miz-1
Authors: Walisko Oliver , Izsvák Zsuzsanna , Szabó Kornélia , Kaufman Christopher D. , Herold Steffi , Ivics Zoltán
Published: March 2006

We used the
Sleeping Beauty
(SB) transposable element as a tool to probe transposon–host cell interactions in vertebrates. The Miz-1 transcription factor was identified as an interactor of the SB transposase in a yeast two-hybrid screen. Through its association with Miz-1, the SB transposase down-regulates cyclin D1 expression in human cells, as evidenced by differential gene expression analysis using microarray hybridization. Down-regulation of cyclin D1 results in a prolonged G
1
phase of the cell cycle and retarded growth of transposase-expressing cells. G
1
slowdown is associated with a decrease of cyclin D1/cdk4-specific phosphorylation of the retinoblastoma protein. Both cyclin D1 down-regulation and the G
1
slowdown induced by the transposase require Miz-1. A temporary G
1
arrest enhances transposition, suggesting that SB transposition is favored in the G
1
phase of the cell cycle, where the nonhomologous end-joining pathway of DNA repair is preferentially active. Because nonhomologous end-joining is required for efficient SB transposition, the transposase-induced G
1
slowdown is probably a selfish act on the transposon’s part to maximize the chance for a successful transposition event.
Metabotropic Ca2+channel-induced Ca2+release and ATP-dependent facilitation of arterial myocyte contraction
Authors: del Valle-Rodríguez Alberto , Calderón Eva , Ruiz Myriam , Ordoñez Antonio , López-Barneo José , Ureña Juan
Published: March 2006

Voltage-gated Ca2+channels in arterial myocytes can mediate Ca2+release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca2+influx. This metabotropic action of Ca2+channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca2+-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPβS, thus suggesting that ATP directly interacts with Ca2+channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca2+channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca2+channel opening, it might mediate elevations of cytosolic Ca2+and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.
Cortistatin, an antiinflammatory peptide with therapeutic action in inflammatory bowel disease
Authors: Gonzalez-Rey Elena , Varela Nieves , Sheibanie Amir F. , Chorny Alejo , Ganea Doina , Delgado Mario
Published: March 2006

Cortistatin is a recently discovered cyclic neuropeptide related to somatostatin that has emerged as a potential endogenous antiinflammatory factor based on its production by, and binding to, immune cells. Crohn’s disease is a chronic debilitating disease characterized by severe T helper 1 (Th1)-driven inflammation of the gastrointestinal tract. The aim of this study is to investigate the therapeutic effect of cortistatin in a murine model of colitis. Cortistatin treatment significantly ameliorated the clinical and histopathologic severity of the inflammatory colitis, abrogating body weight loss, diarrhea, and inflammation and increased the survival rate of the colitic mice. The therapeutic effect was associated with down-regulation of inflammatory and Th1-driven autoimmune response, including the regulation of a wide spectrum of inflammatory mediators. In addition, a partial involvement of regulatory IL-10-secreting T cells in this therapeutic effect was demonstrated. Importantly, cortistatin treatment was therapeutically effective in established colitis and avoided the recurrence of the disease. This work identifies cortistatin as an antiinflammatory factor with the capacity to deactivate the intestinal inflammatory response and restore mucosal immune tolerance at multiple levels. Consequently, cortistatin represents a multistep therapeutic approach for the treatment of Crohn’s disease and other Th1-mediated inflammatory diseases.
Identification of a genetic cluster influencing memory performance and hippocampal activity in humans
Authors: de Quervain Dominique J.-F. , Papassotiropoulos Andreas
Published: March 2006

Experimental work in animals has shown that memory formation depends on a cascade of molecular events. Here we show that variability of human memory performance is related to variability in genes encoding proteins of this signaling cascade, including the NMDA and metabotrobic glutamate receptors, adenylyl cyclase, CAMKII, PKA, and PKC. The individual profile of genetic variability in these signaling molecules correlated significantly with episodic memory performance (
P
< 0.00001). Moreover, functional MRI during memory formation revealed that this genetic profile correlated with activations in memory-related brain regions, including the hippocampus and parahippocampal gyrus. The present study indicates that genetic variability in the human homologues of memory-related signaling molecules contributes to interindividual differences in human memory performance and memory-related brain activations.
Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray
Authors: Zhu Heng , Hu Shaohui , Jona Ghil , Zhu Xiaowei , Kreiswirth Nate , Willey Barbara M. , Mazzulli Tony , Liu Guozhen , Song Qifeng , Chen Peng , Cameron Mark , Tyler Andrea , Wang Jian , Wen Jie , Chen Weijun , Compton Susan , Snyder Michael
Published: March 2006

To monitor severe acute respiratory syndrome (SARS) infection, a coronavirus protein microarray that harbors proteins from SARS coronavirus (SARS-CoV) and five additional coronaviruses was constructed. These microarrays were used to screen ≈400 Canadian sera from the SARS outbreak, including samples from confirmed SARS-CoV cases, respiratory illness patients, and healthcare professionals. A computer algorithm that uses multiple classifiers to predict samples from SARS patients was developed and used to predict 206 sera from Chinese fever patients. The test assigned patients into two distinct groups: those with antibodies to SARS-CoV and those without. The microarray also identified patients with sera reactive against other coronavirus proteins. Our results correlated well with an indirect immunofluorescence test and demonstrated that viral infection can be monitored for many months after infection. We show that protein microarrays can serve as a rapid, sensitive, and simple tool for large-scale identification of viral-specific antibodies in sera.
Global asymptotic coherence in discrete dynamical systems
Authors: Earn David J. D. , Levin Simon A.
Published: March 2006

Spatial synchrony (coherence) in dynamical systems is of both theoretical and applied importance. We address this problem for a generalization of coupled map lattices (CMLs). In the systems we study, which we term “meta-CMLs,” the map at each lattice point may be multidimensional (corresponding, for example, to multispecies ecological systems in which all species have the same dispersal pattern). Most previous work on coherence of CMLs has focused on local stability. Here, we prove a global theorem that provides a useful sufficient condition guaranteeing decay of incoherence in meta-CMLs regardless of initial conditions and regardless of the nature of the attractors of the system. This result facilitates useful analyses of a variety of applied problems, including conservation of endangered species and eradication of pests or infectious diseases.
The 3D profile method for identifying fibril-forming segments of proteins
Authors: Thompson Michael J. , Sievers Stuart A. , Karanicolas John , Ivanova Magdalena I. , Baker David , Eisenberg David
Published: March 2006

Based on the crystal structure of the cross-β spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed β-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using
rosettadesign
, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of ≈80% with a
P
value of ≈10
−12
when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.
Mechanism of Akt1 inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2
Authors: Liu Hong , Radisky Derek C. , Nelson Celeste M. , Zhang Hui , Fata Jimmie E. , Roth Richard A. , Bissell Mina J.
Published: March 2006

Akt1 is frequently up-regulated in human tumors and has been shown to accelerate cell proliferation and to suppress programmed cell death; consequently, inhibition of the activity of Akt1 has been seen as an attractive target for therapeutic intervention. Paradoxically, hyperactivation of the Akt1 oncogene can also prevent the invasive behavior that underlies progression to metastasis. Here we show that overexpression of activated myr-Akt1 in human breast cancer cells phosphorylates and thereby targets the tumor suppressor tuberous sclerosis complex 2 (TSC2) for degradation, leading to reduced Rho-GTPase activity, decreased actin stress fibers and focal adhesions, and reduced motility and invasion. Overexpression of TSC2 rescues the migration phenotype of myr-Akt1-expressing tumor cells, and high levels of TSC2 in breast cancer patients correlate with increased metastasis and reduced survival. These data indicate that the functional properties of genes designated as oncogenes or tumor suppressor genes depend on the context of the cell type and the tissues studied, and suggest the need for caution in designing therapies targeting the function of individual genes in epithelial tissues.
Quasiregular mappings from a punctured ball into compact manifolds
Authors: Pankka Pekka
Published: March 2006

We study quasiregular mappings from a punctured unit ball of the Euclideannn-space into compact manifolds. We show that a quasiregular mapping has a limit in the point of punctuation whenever the dimension of the cohomology ring of the compact manifold exceeds a bound given in terms of the dimension and the distortion constant of the mapping.
Rac3-induced Neuritogenesis Requires Binding to Neurabin I
Authors: Orioli Donata , Colaluca Ivan N. , Stefanini Miria , Riva Silvano , Dotti Carlos G. , Peverali Fiorenzo A.
Published: March 2006

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.
A calibration method for estimating absolute expression levels from microarray data
Authors: Engelen Kristof , Naudts Bart , De Moor Bart , Marchal Kathleen
Published: March 2006

Motivation: We describe an approach to normalize spotted microarray data, based on a physically motivated calibration model. This model consists of two major components, describing the hybridization of target transcripts to their corresponding probes on the one hand, and the measurement of fluorescence from the hybridized, labeled target on the other hand. The model parameters and error distributions are estimated from external control spikes.

Results: Using a publicly available dataset, we show that our procedure is capable of adequately removing the typical non-linearities of the data, without making any assumptions on the distribution of differences in gene expression from one biological sample to the next. Since our model links target concentration to measured intensity, we show how absolute expression values of target transcripts in the hybridization solution can be estimated up to a certain degree.

Contact:  kathleen.marchal@biw.kuleuven.be

Supplementary information: Supplementary data are available at Bioinformatics online.
Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response
Authors: Becker Emmanuelle , Meyer Vincent , Madaoui Hocine , Guerois Raphaël
Published: March 2006

Motivation: Human Nbs1 and its homolog Xrs2 in Saccharomyces cerevisiae are part of the conserved MRN complex (MRX in yeast) which plays a crucial role in maintaining genomic stability. NBS1 corresponds to the gene mutated in the Nijmegen breakage syndrome (NBS) known as a radiation hyper-sensitive disease. Despite the conservation and the importance of the MRN complex, the high sequence divergence between Nbs1 and Xrs2 precluded the identification of common domains downstream of the N-terminal Fork-Head Associated (FHA) domain.

Results: Using HMM–HMM profile comparisons and structure modelling, we assessed the existence of a tandem BRCT in both Nbs1 and Xrs2 after the FHA. The structure-based conservation analysis of the tandem BRCT in Nbs1 supports its function as a phosphoserine binding domain. Remarkably, the 5 bp deletion observed in 95% of NBS patients cleaves the tandem at the linker region while preserving the structural integrity of each BRCT domain in the resulting truncated gene products.

Contact:  guerois@cea.fr

Supplementary information:
Convergence of the proteomic pattern in cancer
Authors: Müller Ute , Ernst Günther , Melle Christian , Guthke Reinhard , von Eggeling Ferdinand
Published: March 2006

Motivation: On the histological level the differentiation of normal epithelial tissues is well known. The phenomenon of dedifferentiation, which occurs as cells develop towards malignancy is also well described. To identify an epithelial tumor-specific proteomic profile as well as to measure the proximities between we used data from tumor tissue and adjacent normal tissue microdissected from head and neck and colon cancer samples which were analyzed using ProteinChip technology and performed a bioinformatic meta-analysis on the resulting four complex datasets.

Results: All four groups could be identified based on their proteomic signatures and the tumor tissues were found to be more similar to one another than to the normal epithelial tissue from which they progressed. This study shows at the proteomic level that changes in the histological features of tumors as compared to the tissues from which they arise are reflected in the convergence of proteomic pattern during the development to cancer.

Contact:  fegg@mti.uni-jena.de

Supplementary information: Supplementary data are available at Bioinformatics online.
Predicting protein interaction sites: binding hot-spots in protein–protein and protein–ligand interfaces
Authors: Burgoyne Nicholas J. , Jackson Richard M.
Published: March 2006

Motivation: Protein assemblies are currently poorly represented in structural databases and their structural elucidation is a key goal in biology. Here we analyse clefts in protein surfaces, likely to correspond to binding ‘hot-spots’, and rank them according to sequence conservation and simple measures of physical properties including hydrophobicity, desolvation, electrostatic and van der Waals potentials, to predict which are involved in binding in the native complex.

Results: The resulting differences between predicting binding-sites at protein–protein and protein–ligand interfaces are striking. There is a high level of prediction accuracy (≤93%) for protein–ligand interactions, based on the following attributes: van der Waals potential, electrostatic potential, desolvation and surface conservation. Generally, the prediction accuracy for protein–protein interactions is lower, with the exception of enzymes. Our results show that the ease of cleft desolvation is strongly predictive of interfaces and strongly maintained across all classes of protein-binding interface.

Contact:  r.m.jackson@leeds.ac.uk

Supplementary information: Supplementary data are available at Bioinformatics online.
MACGT: multi-dimensional automated clustering genotyping tool for analysis of microarray-based mini-sequencing data
Authors: Walley David C. , Tripp Ben W. , Song Young C. , Walley Keith R. , Tebbutt Scott J.
Published: March 2006

Summary: Multi-dimensional Automated Clustering Genotyping Tool (MACGT) is a Java application that clusters complex multi-dimensional vector data derived from single nucleotide polymorphism (SNP) genotyping experiments using mini-sequencing based microarray chemistries such as arrayed primer extension (APEX). Spot intensity output files from microarray experiments across multiple samples are imported into MACGT. The datasets can include four channels of intensity data for each spot, replica spots for each SNP probe and multiple probe types (APEX and allele-specific APEX probes) on both DNA strands for each SNP. MACGT automatically clusters these multi-dimensionality datasets for each SNP across multiple samples. Incorporation of additional array datasets from known samples that have previously validated SNP genotype calls allows unknown samples to be automatically assigned a genotype based on the clustering, along with numerical measures of confidence for each genotype call. Calling accuracy by MACGT exceeds 98% when applied to genotyping data from APEX microarrays, and can be increased to >99.5% by applying thresholds to the confidence measures.

Availability: MACGT is open source and is freely available (under a GNU General Public License) from the iCAPTURE Centre web site, .

Contact:  stebbutt@mrl.ubc.ca

Supplementary information: Additional information, including Supplementary Figure S1, test data and a user's manual, is available from the iCAPTURE Centre web site (see above).
AGRIS and AtRegNet. A Platform to Link cis-Regulatory Elements and Transcription Factors into Regulatory Networks
Authors: Palaniswamy Saranyan K. , James Stephen , Sun Hao , Lamb Rebecca S. , Davuluri Ramana V. , Grotewold Erich
Published: March 2006

Gene regulatory pathways converge at the level of transcription, where interactions among regulatory genes and between regulators and target genes result in the establishment of spatiotemporal patterns of gene expression. The growing identification of direct target genes for key transcription factors (TFs) through traditional and high-throughput experimental approaches has facilitated the elucidation of regulatory networks at the genome level. To integrate this information into a Web-based knowledgebase, we have developed the Arabidopsis Gene Regulatory Information Server (AGRIS). AGRIS, which contains all Arabidopsis (Arabidopsis thaliana) promoter sequences, TFs, and their target genes and functions, provides the scientific community with a platform to establish regulatory networks. AGRIS currently houses three linked databases: AtcisDB (Arabidopsis thaliana cis-regulatory database), AtTFDB (Arabidopsis thaliana transcription factor database), and AtRegNet (Arabidopsis thaliana regulatory network). AtTFDB contains 1,690 Arabidopsis TFs and their sequences (protein and DNA) grouped into 50 (October 2005) families with information on available mutants in the corresponding genes. AtcisDB consists of 25,806 (September 2005) promoter sequences of annotated Arabidopsis genes with a description of putative cis-regulatory elements. AtRegNet links, in direct interactions, several hundred genes with the TFs that control their expression. The current release of AtRegNet contains a total of 187 (September 2005) direct targets for 66 TFs. AGRIS can be accessed at http://Arabidopsis.med.ohio-state.edu.
Identification of correct regions in protein models using structural, alignment, and consensus information
Authors: Wallner Björn , Elofsson Arne
Published: March 2006

In this study we present two methods to predict the local quality of a protein model: ProQres and ProQprof. ProQres is based on structural features that can be calculated from a model, while ProQprof uses alignment information and can only be used if the model is created from an alignment. In addition, we also propose a simple approach based on local consensus, Pcons‐local. We show that all these methods perform better than state‐of‐the‐art methodologies and that, when applicable, the consensus approach is by far the best approach to predict local structure quality. It was also found that ProQprof performed better than other methods for models based on distant relationships, while ProQres performed best for models based on closer relationship, i.e., a model has to be reasonably good to make a structural evaluation useful. Finally, we show that a combination of ProQprof and ProQres (ProQlocal) performed better than any other nonconsensus method for both high‐ and low‐quality models. Additional information and Web servers are available at:http://www.sbc.su.se/∼bjorn/ProQ/.
Solvation of the folding‐transition state in Pseudomonas aeruginosa azurin is modulated by metal
Authors: Wilson Corey J. , Apiyo David , Wittung‐Stafshede Pernilla
Published: March 2006

The role of water in protein folding, specifically its presence or not in the transition‐state structure, is an unsolved question. There are two common classes of folding‐transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (φ) values, and polarized transition states, which instead display distinct substructures with very high φ‐values. Apo‐and zinc‐forms of Pseudomonas aeruginosa azurin both fold in two‐state equilibrium and kinetic reactions; while the apo‐form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding‐transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic‐to‐alanine φ‐value analysis, valine‐to‐threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding‐transition state upon combination with regular φ‐values. We find that the transition state for folding of apo‐azurin appears completely dry, while that for zinc‐azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo‐and holo‐folding nuclei can be rationalized in terms of the shape of the free‐energy barrier.
New Escherichia coli outer membrane proteins identified through prediction and experimental verification
Authors: Marani Paola , Wagner Samuel , Baars Louise , Genevaux Pierre , De Gier Jan‐Willem , Nilsson Ingmarie , Casadio Rita , Von Heijne Gunnar
Published: March 2006

Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild‐type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five—YftM, YaiO, YfaZ, CsgF, and YliI—and also provide preliminary data indicating that a sixth—YfaL—may be an outer membrane autotransporter.
Cofactor effects on the protein folding reaction: Acceleration of α‐lactalbumin refolding by metal ions
Authors: Bushmarina Natalia A. , Blanchet Clément E. , Vernier Grégory , Forge Vincent
Published: March 2006

About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with α‐lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of α‐lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady‐state data in association with the Φ value method indicates the binding of the metal ions on the unfolded state of α‐lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.
Dynamic motions of free and bound Ø29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry
Authors: Fu Chi‐Yu , Prevelige Peter E.
Published: March 2006

In the double‐stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid‐bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N‐terminal helix‐loop‐helix (H‐L‐H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid‐bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.
NMR study of the tetrameric KcsA potassium channel in detergent micelles
Authors: Chill Jordan H. , Louis John M. , Miller Christopher , Bax Ad
Published: March 2006

Nuclear magnetic resonance (NMR) studies of large membrane‐associated proteins are limited by the difficulties in preparation of stable protein–detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68‐kDa homotetrameric KcsA, a thermostable N‐terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C‐terminal cytoplasmic domain, absent in the original structure, contains a 14‐residue helix that could participate in tetramerization by forming an intersubunit four‐helix bundle. A quantitative estimate of cross‐ relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS–KcsA mixed micelles form an oblate spheroid with ∼180 SDS molecules per channel. K+ ions bind to the micelle‐solubilized channel with a KD of 3 ± 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH‐induced changes in chemical shift along the “outer” transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH‐gating of the potassium channel.
Evolutionary links between FliH/YscL‐like proteins from bacterial type III secretion systems and second‐stalk components of the FoF1 and vacuolar ATPases
Authors: Pallen Mark J. , Bailey Christopher M. , Beatson Scott A.
Published: March 2006

Bacterial type III secretion drives flagellar biosynthesis and mediates bacterial‐eukaryotic interactions. Type III secretion is driven by an ATPase that is homologous to the catalytic subunits of proton‐translocating ATPases, such as the FoF1 ATPase. Here we use PSI‐BLAST searches to show that some noncalatytic components are also conserved between type III secretion systems and proton‐translocating ATPases. In particular, we show that the FliH/YscL‐like proteins and the E subunits of vacuolar ATPases represent fusions of domains homologous to second‐stalk components of the FoF1 ATPase (the b and δ subunits).
Crystal structure of 3‐hydroxyanthranilic acid 3,4‐dioxygenase from Saccharomyces cerevisiae: A special subgroup of the type III extradiol dioxygenases
Authors: Li Xiaowu , Guo Min , Fan Jun , Tang Wenying , Wang Deqiang , Ge Honghua , Rong Hui , Teng Maikun , Niu Liwen , Liu Qun , Hao Quan
Published: March 2006

3‐Hydroxyanthranilic acid 3,4‐dioxygenase (3HAO) is a non‐heme ferrous extradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzes the conversion of 3‐hydroxyanthranilate (HAA) to quinolinic acid (QUIN), an endogenous neurotoxin, via the activation of N‐methyl‐D‐aspartate (NMDA) receptors and the precursor of NAD+ biosynthesis. The crystal structure of 3HAO from S. cerevisiae at 2.4 Å resolution shows it to be a member of the functionally diverse cupin superfamily. The structure represents the first eukaryotic 3HAO to be resolved. The enzyme forms homodimers, with two nickel binding sites per molecule. One of the bound nickel atoms occupies the proposed ferrous‐coordinated active site, which is located in a conserved double‐strand β‐helix domain. Examination of the structure reveals the participation of a series of residues in catalysis different from other extradiol dioxygenases. Together with two iron‐binding residues (His49 and Glu55), Asp120, Asn51, Glu111, and Arg114 form a hydrogen‐bonding network; this hydrogen‐bond network is key to the catalysis of 3HAO. Residues Arg101, Gln59, and the substrate‐binding hydrophobic pocket are crucial for substrate specificity. Structure comparison with 3HAO from Ralstonia metallidurans reveals similarities at the active site and suggests the same catalytic mechanism in prokaryotic and eukaryotic 3HAO. Based on sequence comparison, we suggest that bicupin of human 3HAO is the first example of evolution from a monocupin dimer to bicupin monomer in the diverse cupin superfamilies. Based on the model of the substrate HAA at the active site of Y3HAO, we propose a mechanism of catalysis for 3HAO.
Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding
Authors: Kotaka Masayo , Dhaliwal Balvinder , Ren Jingshan , Nichols Charles E. , Angell Richard , Lockyer Michael , Hawkins Alastair R. , Stammers David K.
Published: March 2006

Methicillin‐resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug‐resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate‐bound forms of S. aureus thymidylate kinase (SaTMK). His‐tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix α3 shifting position initially, followed by movement of α2 to close the substrate site. In addition, we observe significant conformational differences in the TMP‐binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP‐binding site, close to the thymine ring, whereas a cis‐proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.
Functional contribution of a conserved, mobile loop histidine of phosphoribulokinase
Authors: Runquist Jennifer A. , Miziorko Henry M.
Published: March 2006

In the Rhodobacter sphaeroides phosphoribulokinase (PRK) structure, there are several disordered regions, including a loop containing invariant residues Y98 and H100. The functional importance of these residues has been unclear. PRK is inactivated by diethyl pyrocarbonate (DEPC) and protected by the substrates ATP and Ru5P, as well as by the competitive inhibitor, 6‐phosphogluconate, suggesting active site histidine residue(s). PRK contains only three invariant histidines: H45, H100, and H134. Previous mutagenesis studies discount significant function for H134, but implicate H45 in Ru5P binding. PRK mutant H45N is inactivated by DEPC, implicating a second active site histidine. To evaluate the function of H100, as well as another invariant loop residue Y98, PRK mutants Y98L, H100A, H100N, and H100Q were characterized. Mutant PRK binding stoichiometries for the fluorescent alternative substrate, trinitrophenyl‐ATP, as well as the allosteric activator, NADH, are comparable to wild‐type PRK values, suggesting intact effector and substrate binding sites. The KmRu5P for the H100 mutants shows modest eight‐ to 14‐fold inflation effects, whereas Y98L exhibits a 40‐fold inflation for KmRu5P. However, Y98L's Ki for the competitive inhibitor 6‐phosphogluconate is close to that of wild‐type PRK. These observations suggest that Y98 and H100 are not essential Ru5P binding determinants. The Vm of Y98L is diminished 27‐fold compared with wild‐type PRK. In contrast, H100A, H100N, and H100Q exhibit significant decreases in Vm of 2600‐, 2300‐, and 735‐fold, respectively. Results suggest that the mobile region containing Y98 and H100 must contribute to PRK's active site. Moreover, H100's imidazole significantly influences catalytic efficiency.
Crystal structure of Homo sapiens PTD012 reveals a zinc‐containing hydrolase fold
Authors: Manjasetty Babu A. , Büssow Konrad , Fieber‐Erdmann Martin , Roske Yvette , Gobom Johan , Scheich Christoph , Götz Frank , Niesen Frank H. , Heinemann Udo
Published: March 2006

The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X‐ray structure analysis at 1.7 Å resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non‐functional. The structure of PTD012 displays an αββα four‐layer topology. A metal ion residing between the central β‐sheets is partially coordinated by three histidine residues. X‐ray absorption near‐edge structure (XANES) analysis identifies the PTD012‐bound ion as Zn2+. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn2+ to PTD012 is reminiscent of zinc‐containing enzymes such as carboxypeptidase, carbonic anhydrase, and β‐lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p‐nitrophenyl acetate.
Antibody targeting of TIRC7 results in significant therapeutic effects on collagen-induced arthritis in mice
Authors: Utku N , Heinemann T , Winter M , Bulwin C G , Schlawinsky M , Fraser P , Nieuwenhuis E E S , Volk H-D , Blumberg R S
Published: March 2006

TIRC7 is a cell surface molecule which is expressed in T and B lymphocytes and negatively regulates their function. Anti-TIRC7 specific monoclonal antibody (mAb) inhibited T cell memory response to recall antigens. Up-regulation of TIRC7 on lymphocytes from joint tissue of patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggested TIRC7 as a novel target to promote anti-inflammatory reaction. Anti-TIRC7 mAb administration significantly inhibited the induction and progression of CIA and the anti-collagen IgG1 and IgG2a antibody response. Combination therapy of anti-TIRC7 mAb and soluble TNF-α receptor demonstrated an increased inhibitory effect over the single compounds on CIA. The results demonstrate the therapeutic potential of TIRC7 targeting with mAb in diseases associated with exaggerated T and B cell responses.
Cerebral leucocyte infiltration in lupus-prone MRL/MpJ-faslpr mice − roles of intercellular adhesion molecule-1 and P-selectin
Authors: James W G , Hutchinson P , Bullard D C , Hickey M J
Published: March 2006

The autoimmune disease which affects MRL/MpJ-faslpr mice results in cerebral leucocyte recruitment and cognitive dysfunction. We have previously observed increased leucocyte trafficking in the cerebral microcirculation of these mice; however, the types of leucocytes recruited have not been analysed thoroughly, and the roles of key endothelial adhesion molecules in recruitment of these leucocytes have not been investigated. Therefore the aim of this study was to classify the phenotypes of leucocytes present in inflamed brains of MRL/MpJ-faslpr mice, and dissect the roles of endothelial adhesion molecules in their accumulation in the brain. Immunohistochemical analysis revealed significant leucocyte infiltration in the brains of 16- and 20-week-old MRL/MpJ-faslpr mice, affecting predominantly the choroid plexus. Isolation of brain-infiltrating leucocytes revealed that lymphocytes and neutrophils were the main populations present. The CD3+ lymphocytes in the brain consisted of similar proportions of CD4+, CD8+ and CD4–/CD8–[double negative (DN)] populations. Assessment of MRL/MpJ-faslpr mice deficient in endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1) or P-selectin indicated that cerebral leucocyte recruitment persisted in the absence of these molecules, with only minor changes in the phenotypes of infiltrating cells. Together these data indicate that the brains of MRL/MpJ-faslpr mice are affected by a mixed leucocyte infiltrate, of which the unusual DN lymphocyte phenotype contributes a substantial proportion. In addition, endothelial adhesion molecules ICAM-1 and P-selectin, which modulate survival of MRL/MpJ-faslpr mice, do not markedly inhibit leucocyte entry into the central nervous system.
The role of Fc-receptors in murine mercury-induced systemic autoimmunity
Authors: Martinsson K , Hultman P
Published: March 2006

Inorganic mercury (Hg) in genetically susceptible mouse strains induces a T cell-dependent, systemic autoimmune condition (HgIA) characterized by immunostimulation, anti-nuclear antibodies (ANA) and systemic immune-complex (IC) deposits. The exact phenotypic expression of HgIA in different strains depends on H-2 and non-H-2 genes. Fc receptors (FcRs) are important in the development of many autoimmune diseases. In this study, the effect of targeted mutations for activating and inhibiting FcRs in the BALB/c model of HgIA was examined. Hg-treated BALB/c mice without mutation (wild-type, wt) showed heavy IC deposits in the renal glomerular mesangium, as well as in renal and splenic vessel walls. The renal mesangial IC deposits were severely reduced in Hg-treated BALB/c mice without the γ-chain (lack of the activating receptors FcγRI, FcγRIII and Fc∈RI), but unchanged in mice lacking the inhibitory FcγRIIB. The Hg-induced vessel wall IC deposits present in wt mice were abolished and reduced in the FcRγ and FcγRIIB strains, respectively. Hg-treated BALB/c wt mice and mice without the γ-chain showed an increase in serum IgE, while the increase in IgG1 was attenuated in the latter strain. In contrast, absence of the inhibiting FcγRIIB augmented the Hg-induced increase of both serum IgG1 and IgE. In conclusion, FcRs are important mainly for the induction of systmeic IC deposits in the HgIA model, but also affects serum IgG1 and IgE levels.
Association of epithelial damage and signs of neutrophil mobilization in the airways during acute exacerbations of paediatric asthma
Authors: Yoshihara S , Yamada Y , Abe T , Lindén A , Arisaka O
Published: March 2006

We examined whether epithelial damage is associated with mobilization of neutrophils or eosinophils in the airway lumen during acute exacerbations of paediatric asthma. Aspirated sputum samples were harvested from 65 paediatric patients (mean age 3·4 ± 0·4 years) during acute exacerbations of asthma. Patients with signs of infection were excluded. The presence of conglomerates of epithelial cells (i.e. ‘Creola bodies) in the aspirated sputum was utilized as a marker of epithelial damage. Among the paediatric asthma patients, 60% displayed Creola bodies (CrB+: n = 39) in their sputum samples whereas the remaining patients did not (CrB–: n = 26). CrB+ patients displayed more than a 20-fold increase in the concentration of the neutrophil-mobilizing cytokine interleukin (IL)-8 (pg/ml) and of the neutrophil product neutrophil elastase (NE, g/l), respectively, compared with CrB– patients (IL-8: 7468·2 ± 1953·6 versus 347·9 ± 72·6, P < 0·01; NE: 2072·4 ± 419·0 versus 438·5 ± 125·7, P < 0·01). Even though not statistically significant, a corresponding trend was observed for the relative number of sputum neutrophils. In contrast, the concentration of the eosinophil-mobilizing cytokine IL-5 and the esoinophil product ECP tended to be lower in CrB+ than in CrB– patients (P > 0·05). In conclusion, as indicated by the analysis of aspirated sputum, epithelial damage is associated with a locally enhanced chemotactic signal for and activity of neutrophils, but not eosinophils, during acute exacerbations of paediatric asthma. It remains to be determined whether these indirect signs of neutrophil mobilization in the airway lumen mirror an increased number of neutrophils in the surrounding airway tissue.
Effects of haemorrhagic hypotension on the subcapsular artery and microvasculature of the rat testis
Authors: Lissbrant E. , Collin O. , Damber J‐E. , Bergh A.
Published: March 2006

Developing germ cells may be sensitive to even moderate reductions in blood flow. Surprisingly, however, experimental evidence suggests that the rat testis may be unable to maintain its blood flow during a decrease in systemic blood pressure. This study was therefore performed in order to answer the following questions: Is the testis able to maintain its blood flow during moderate to major reductions in blood pressure and, if so, at which level of the testicular vasculature (main artery or microcirculation) does this compensatory response take place? Moderate (−20%) and major (−40%) reductions in blood pressure were induced in anaesthetized rats by haemorrhage and the effects on testicular microvascular blood flow and subcapsular testicular artery diameter were examined by using laser Doppler flowmetry and in vivo video‐microscopy respectively. Haemorrhagic hypotension led to decreased local testicular blood flow, but the relative reductions in flow were generally only half as large as the reductions in blood pressure. Hypotension also decreased the diameter of the main subcapsular testicular artery. During large reductions in blood pressure the subcapsular testicular artery constricts and testicular blood flow decreases. However, blood flow is reduced proportionally less than the mean arterial pressure, suggesting that local regulatory mechanisms are present in the testicular microvasculature, which may prevent blood flow from falling below a critical level.
Sexual behaviour, sperm quantity and quality after short‐term streptozotocin‐induced hyperglycaemia in rats
Authors: Scarano W. R. , Messias A. G. , Oliva S. U. , Klinefelter G. R. , Kempinas W. G.
Published: March 2006

Studies of diabetes mellitus in the streptozotocin rat model suggest that sexual dysfunctions may result from diabetes‐induced alterations of the neuroendocrine‐reproductive tract axis. Our investigation was performed to better define the effects of short‐term hyperglycaemia on rat epididymal sperm quantity, quality and transit time, using both natural mating and artificial in utero insemination protocols. Male rats were made diabetic with streptozotocin (sc, 40 mg/kg), whereas controls received vehicle. Sexual behaviour was tested after 15 days and sperm fertilizing ability was checked 22 days after the injection through natural mating and artificial in utero insemination. Other parameters such as daily sperm production, testosterone levels, as well as sperm morphology and motility were also investigated. Fifty per cent of the diabetic animals showed no copulatory behaviour during tests and the number of animals reaching ejaculation was smaller in the diabetic group when compared with the control group (33% vs. 83%). Diabetes resulted in decreased body and reproductive organ weights, as well as diminished sperm counts in the testis and epididymis, that were associated with diminution of plasmatic testosterone levels. After natural mating, there was a decrease in the fertility in the diabetic adult male rats (25.5%) compared with control animals (81.5%). However, distal cauda epididymal sperm from diabetic rats displayed normal fertilization ability (91.5%) using in utero insemination. There were no effects of hyperglycaemia on sperm transit time in the epididymis and on spermatogenesis. Our results indicate that diabetes mellitus produces reproductive dysfunction, but does not compromise sperm fertilizing ability in the cauda epididymis in this experimental model.