bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of cytoplasmic granules imaged by the real‐time microscope, Nile Red and Filipin in fibroblasts from patients with lipid storage diseases
Authors: Pham N.‐A. , Gal M. R. , Bagshaw R. D. , Mohr A. J. , Chue B. , Richardson T. , Callahan J. W.
Published: January 2006

Cytoplasmic granules in fibroblasts, visualized without stains, or labelled with Nile red, Filipin, or anti‐LAMP‐1 (lysosome‐associated membrane protein 1), were imaged using the real‐time microscope (RTM). New advances in light microscope technology were applied to detect cytoplasmic granules (RTM‐visible granules) and characterize them by imaging contrast, size, shape, cellular distribution, composition, motion dynamics and quantity. Appearing as solid spheroids or ring structures, the majority of the RTM‐visible granules contained Nile‐red labelled neutral lipids. A smaller subpopulation, appearing dimmer, with less imaging contrast, contained Filipin‐labelled free cholesterol. Most lipid storage granules have a diameter ranging from 0.3 μm to 0.6 μm, with a small population measuring up to 1 μm. They typically clustered in the perinuclear region and displayed relatively small oscillatory motion. Immunofluorescence based on LAMP‐1 labelling highlighted granular structures that were distinct and separate from RTM‐visible granules and other structures in the light modality of the microscope. RTM‐visible granules were associated with disease phenotypes that have increased cellular neutral lipid stores corresponding to the Nile red‐labelled droplets (e.g. triacylglycerides, cholesterol esters). As predicted, the fibroblast strains with a defect resulting in Wolman disease, when compared to control samples, consistently had RTM‐visible granules, higher in imaging contrast and with larger diameters, that were labelled with Nile red, and also an increased frequency of Filipin–cholesterol complexes. By comparison, in fibroblasts where the lipid storage is less evident (Gaucher and Farber diseases) or from GM1 gangliosidosis, where the primary storage substances are oligosaccharides, fewer and smaller RTM‐visible granules were observed. In some cases, changes in contrast and morphology in the unstained cytoplasmic compartments were more evident than in the labelled structures. In summary, applying the RTM imaging system to fibroblasts enables differences between the various disease types to be seen and, in specific examples, a unique phenotype can be readily discerned.
Comprehensive analysis of pyrimidine metabolism in 450 children with unspecific neurological symptoms using high–pressure liquid chromatography–electrospray ionization tandem mass spectrometry
Authors: Schmidt C. , Hofmann U. , Kohlmüller D. , Mürdter T. , Zanger U. M. , Schwab M. , Hoffmann G. F.
Published: January 2006

To evaluate the significance of inborn metabolic disorders of the pyrimidine degradation pathway, 450 children with unspecific neurological symptoms were comprehensively studied; 200 healthy children were recruited as controls. Uracil and thymine as well as their degradation products in urine were determined with an improved method based on reversed‐phase HPLC coupled with electrospray ionization tandem mass spectrometry and detection by multiple‐reaction monitoring using stable‐isotope‐labelled reference compounds as internal standards.

From the results of the control group we established age‐related reference ranges of all pyrimidine degradation products. In the patient group, two children with dihydropyrimidine dehydrogenase (DPYD) deficiency were identified; one of these was homozygous for the exon 14‐skipping mutation of the DPYD gene. In addition, two patients with high uracil, dihydrouracil and β‐ureidopropionate were found to have ornithine transcarbamylase deficiency. In the urine of 9 patients, β‐alanine was markedly elevated owing to treatment with vigabatrin, an irreversible inhibitor of GABA transaminase, which interferes with β‐alanine breakdown. Four patients had exclusively high levels of β‐aminoisobutyrate (β‐AIB) due to a low activity of the D‐β‐AIB‐pyruvate aminotransferase, probably without clinical significance.

In conclusion, quantitative investigation of pyrimidine metabolites in children with unexplained neurological symptoms, particularly epileptic seizures with or without psychomotor retardation, can be recommended as a helpful tool for diagnosis in clinical practice. Sensitive methods and age‐related reference ranges enable the detection of partial enzyme deficiencies.
Gordon syndrome and succinylcholine
Authors: Puura A. , Schultz R.
Published: January 2006

We present a case of 6‐year‐old boy who developed severe hyperkalaemia and ventricular tachycardia after administration of succinylcholine. Ventricular tachycardia was defibrillated and Gordon syndrome, which was diagnosed subsequently, was treated successfully. Paediatric patients with underlying metabolic disorders such as Gordon syndrome are at high risk of developing severe hyperkalaemia after succinylcholine administration.
Mutation spectrum of type I glycogen storage disease in Hungary
Authors: Miltenberger‐Miltenyi G. , Szonyi L. , Balogh L. , Utermann G. , Janecke A. R.
Published: January 2006

We performed mutation analysis in 12 Hungarian type I glycogen storage disease (GSD I) patients in order to determine the mutation spectrum. All patients were clinically classified as GSD Ia. Nine patients carried biallelic G6PC mutations (p.Q27fsX35, p.D38V, p.W70X, p.K76N, p.W77R, p.R83C, p.E110Q, p.G222R), with E110Q reported only in Hungary. However, three patients displayed two common G6PT1 (SLC37A4) mutations (p.L348fsX400, p.C183R) which were originally described in association with GSD Inon‐a. Review of the literature and our data show that G6PT1 mutations are not associated with neutropenia and related clinical findings in approximately 10% of these cases. Homozygosity for the truncating G6PT1 mutation p.L348fsX400 can be observed with and without neutropenia, indicating that one or more modifiers of the action of G6PT1 exist. Our data are suitable to provide DNA‐based and thus noninvasive confirmation of diagnosis in Hungarian patients with this disorder.
Mutational spectrum in ten Italian patients affected by methylmalonyl‐CoA mutase deficiency
Authors: Cavicchi C. , Donati M. A. , Pasquini E. , Poggi G. M. , Dionisi‐Vici C. , Parini R. , Zammarchi E. , Morrone A.
Published: January 2006

We report seven novel mutations, including three amino acids substitutions (p.Glu286Lys, p.Cys560Tyr, p.Pro615Leu), two nonsense mutations (p.Arg31X, p.Glu 451X), one splicing defect (c.2125−1G >A), one small deletion (c.1758–1759delA) and nine previously described mutations identified in 10 unrelated Italian patients affected by mut MMA.
Hyperammonaemic encephalopathy induced by a commercial very‐low‐energy diet in a neglected ornithine‐carbamoyltransferase‐deficient woman
Authors: Vinzio S. , Perrin A. E. , Forestier E. , Schlienger J. L. , Goichot B.
Published: January 2006

This case report describes a 31‐year‐old woman with a neglected ornithine carbamoyltransferase deficiency, admitted for a hyperammonaemic encephalopathy induced by a commercial very low‐energy diet. To our knowledge, this is the first reported case and it underlines the potential risk of such a diet in patients with undiagnosed or neglected ornithine carbamoyltransferase deficiency.
Mucopolysaccharidosis I under enzyme replacement therapy with laronidase—A mortality case with autopsy report
Authors: Lin H.‐Y. , Lin S.‐P. , Chuang C.‐K. , Chen M.‐R. , Chen B.‐F. , Wraith J. E.
Published: January 2006

There is little information about MPS I‐related complications during laronidase therapy. We describe the first autopsy report of a young male MPS I patient who died of infection‐induced cardiopulmonary failure following 2 years of weekly treatment with laronidase.
Methionine adenosyltransferase (MAT) I/III deficiency with concurrent hyperhomocysteinaemia: Two novel cases
Authors: Linnebank M. , Lagler F. , Muntau A. C. , Röschinger W. , Olgemöller B. , Fowler B. , Koch H. G.
Published: January 2006

This study reports three novel mutations of the methionine adenosyltransferase (MAT) lA gene and confirms that hyperhomocysteinaemia may be a characteristic finding in MAT I/III deficiency. Thus, MAT I/III deficiency is important in the differential diagnoses of hyperhomocysteinaemia, which may lead to clinical complications of MAT I/III deficiency.
Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate
Authors: Walker Glenn , Cai Fan , Shen Peiyan , Reynolds Coralie , Ward Brent , Fone Christopher , Honda Shuji , Koganei Megumi , Oda Munehiro , Reynolds Eric
Published: January 2006

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.
Variable quality compression of fluid dynamical data sets using a 3‐D DCT technique
Authors: Loddoch Alexander , Schmalzl Jörg
Published: January 2006

In this paper we present a data compression scheme that is especially suited for the compression of data sets resulting from computational fluid dynamics (CFD). By adopting the concept of the JPEG image compression standard and extending the approach of Schmalzl (2003), we employ a three‐dimensional discrete cosine transform of the data. The resulting frequency components are rearranged, quantized, and, finally, stored using standard variable length integer codes. The compression ratio and also the introduced loss of accuracy can be adjusted over a wide range by means of two compression parameters to give the desired compression profile. Using the proposed technique, compression ratios of more than 60:1 are possible with a mean error of the compressed data of less than 0.05%.
An analysis of statistical characteristics of stratus and stratocumulus over eastern Pacific
Authors: Zhou Mingyu , Zeng Xubin , Brunke Michael , Zhang Zhanhai , Fairall Chris
Published: January 2006

Observational measurements of cloud base, top, thickness, fraction, and liquid water path (LWP) are analyzed to study the macro‐ and micro‐physical properties of stratus and stratocumulus over the eastern Pacific. It is found that the increase in liquid water content with height in cloud is significantly less than the adiabatic rate. The variations of cloud base and top both contribute to the variation of cloud thickness that approximately fits a normal distribution better than either cloud base or top. Because the distribution of LWP depends on the intervals of cloud fraction (or other environmental variables, for example, relative humidity), the determination of cloud fraction from the mean LWP alone would introduce large uncertainties particularly when LWP is large.
Snowfall in coastal West Antarctica much greater than previously assumed
Authors: van den Broeke Michiel , van de Berg Willem Jan , van Meijgaard Erik
Published: January 2006

A new Antarctic accumulation distribution, based on regional model output calibrated with 1900 in‐situ observations, is used to re‐assess accumulation in 24 Antarctic ice drainage basins. When compared to the previous compilation, good agreement is found for 19 of the 24 basins, representing 93% of the ice sheet that is reasonably well covered with observations. In contrast, the Amundsen Sea sector of West Antarctica and the western Antarctic Peninsula, both data sparse regions, are found to receive 80–96% more accumulation than previously assumed. For the Pine Island and Thwaites Glaciers (West Antarctica), which have recently undergone rapid acceleration and thinning, this means a downward adjustment of their contribution to global sea level rise from 0.24 to 0.14 mm per year. Model time series do not show a significant change in Antarctic accumulation over the period 1980–2004.
Radiation fluxes and canopy transmittance: Models and measurements inside a willow canopy
Authors: Mõttus Matti , Sulev Madis
Published: January 2006

Upward and downward fluxes of photosynthetically active and broadband solar radiation, and the transmittance of direct solar radiation measured at different heights inside a willow (Salix viminalis) canopy are compared with modeling results. Two approaches are used for modeling the radiation field inside the canopy: solving the radiative transfer equation using the exact kernel discrete ordinates method and a Monte Carlo model. The measured and modeled canopies include a 1‐year‐old willow coppice with leaf area index (LAI) = 5 and a 4‐year‐old coppice with LAI = 8. Biometrical data used in the models are taken from detailed measurements of canopy structure; measured fluxes are used as boundary conditions. An attempt is made to include the effect of clumping on the penetration of direct solar radiation into the radiative transfer model. All models agreed well with measurement results; enhancement of modeled fluxes due to inclusion of clumping depended on the height inside the canopy while the distribution of leaf inclination angle had a minimal effect on the radiation regime of the stand.
Estrogens and Experimental Ischemic Stroke: A Systematic Review
Authors: Gibson Claire L , Gray Laura J , Murphy Sean P , Bath Philip MW
Published: January 2006

Estrogens are believed to provide females with endogenous protection against cerebrovascular events although clinical trials studying long-term hormone replacement have yielded disappointing results. In contrast, estrogens might be neuroprotective after experimental ischemia. We performed a systematic review of controlled experimental studies that administered estrogens before, or after, cerebral ischemia and measured lesion volume. Relevant studies were found from searching PubMed, Embase and Web of Science. From 161 identified publications, 27 studies using 1304 experimental subjects were analyzed using the Cochrane Review Manager software. Estrogens reduced lesion volume in a dose-dependent manner, after either transient ( P < 0.001) or permanent ( P < 0.001) ischemia and whether administered before or up to 4 h after ischemia onset; no studies assessed efficacy for later time periods. The effect size for estrogens decreased with increasing quality scores for studies of transient ischemia. Estrogens reduced lesion volume when administered to ovariectomized females and young adult males, but had no effect in intact females. Limited data were present for aged animals and the full dose-response relationship was not available in all experimental groups. On the basis of these data, estrogens are a candidate treatment for ischemic stroke, although further preclinical studies are also warranted.
Orientation polarization from faster motions in the ultraviscous and glassy diethyl phthalate and its entropy
Authors: Power G. , Vij J. K. , Johari G. P.
Published: January 2006

Dielectric spectra of the β relaxation in glassy and ultraviscous liquid diethyl phthalate show that its relaxation strength Δϵβ, the distribution of times, and the relaxation rate are more sensitive to temperature T in the ultraviscous liquid than in the glassy state. The Δϵβ against temperature plot has an elbow-shaped break near Tg of ∼181K, which is remarkably similar to that observed in the entropy, enthalpy, and volume against temperature plots, and in the plot of Δϵβ against the liquid’s entropy minus its 0K value. The ratio of Δϵβ to diethyl phthalate’s entropy, after subtracting the 0K value, is 1.08×10−3molK∕J in the glassy state at 120.4K, which decreases slowly to 0.81×10−3molK∕J at 176K near Tg and thereafter rapidly increases to 1.57×10−3molK∕J at 190K. Variation in Δϵβ parallels the variation of the entropy. A change in the activation energy of the β process at T>Tg indicates that its rate is also determined by the structure of the ultraviscous liquid. Features of β relaxation are consistent with localized motions of molecules and may not involve small-angle motions of all molecules.
The limitations of Slater’s element-dependent exchange functional from analytic density-functional theory
Authors: Zope Rajendra R. , Dunlap Brett I.
Published: January 2006

Our recent formulation of the analytic and variational Slater-Roothaan (SR) method, which uses Gaussian basis sets to variationally express the molecular orbitals, electron density, and the one-body effective potential of density-functional theory, is reviewed. Variational fitting can be extended to the resolution of identity method, where variationality then refers to the error in each two-electron integral and not to the total energy. However, a Taylor-series analysis shows that all analytic ab initio energies calculated with variational fits to two-electron integrals are stationary. It is proposed that the appropriate fitting functions be charge neutral and that all ab initio energies be evaluated using two-center fits of the two-electron integrals. The SR method has its root in Slater’s Xα method and permits an arbitrary scaling of the Slater-Gàspàr-Kohn-Sham exchange-correlation potential around each atom in the system. The scaling factors are Slater’s exchange parameters α. Of several ways of choosing these parameters, two most obvious are the Hartree-Fock (HF) αHF values and the exact atomic αEA values. The former are obtained by equating the self-consistent Xα energy and the HF energies, while the latter set reproduces exact atomic energies. In this work, we examine the performance of the SR method for predicting atomization energies, bond distances, and ionization potentials using the two sets of α parameters. The atomization energies are calculated for the extended G2 set of 148 molecules for different basis-set combinations. The mean error (ME) and mean absolute error (MAE) in atomization energies are about 25 and 33kcal∕mol, respectively, for the exact atomic αEA values. The HF values of exchange parameters αHF give somewhat better performance for the atomization energies with ME and MAE being about 15 and 26kcal∕mol, respectively. While both sets give performance better than the local-density approximation or the HF theory, the errors in atomization energy are larger than the target chemical accuracy. To further improve the performance of the SR method for atomization energies, a new set of α values is determined by minimizing the MAE in atomization energies of 148 molecules. This new set gives atomization energies half as large (MAE ∼14.5kcal∕mol) and that are slightly better than those obtained by one of the most widely used generalized-gradient approximations. Further improvements in atomization energies require going beyond Slater’s functional form for exchange employed in this work to allow exchange-correlation interactions between electrons of different spins. The MAE in ionization potentials of 49 atoms and molecules is about 0.5 eV and that in bond distances of 27 molecules is about 0.02 Å. The overall good performance of the computationally efficient SR method using any reasonable set of α values makes it a promising method for study of large systems.
Singular solutions to the Seiberg-Witten and Freund equations on flat space from an iterative method
Authors: Mosna Ricardo A.
Published: January 2006

Although it is well known that the Seiberg-Witten equations do not admit nontrivial L2 solutions in flat space, singular solutions to them have been previously exhibited—either in R3 or in the dimensionally reduced spaces R2 and R1—which have physical interest. In this work, we employ an extension of the Hopf fibration to obtain an iterative procedure to generate particular singular solutions to the Seiberg-Witten and Freund equations on flat space. Examples of solutions obtained by such method are presented and briefly discussed.
Intelligent potentiostat for identification of heavy metals in situ
Authors: Christidis K. , Gow K. , Robertson P. , Pollard P.
Published: January 2006

This article presents a low-cost portable electrochemical instrument capable of on-site identification of heavy metals. The instrument acquires metal-specific voltage and current signals by the application of differential pulse anodic stripping voltammetry. This technique enhances the analytical current and rejects the background current, resulting in a higher signal-to-noise ratio for a better detection limit. The identification of heavy metals is based on an intelligent machine-based method using a multilayer perceptron neural network consisting of three layers of neurons. The neural network is implemented using a 16 bit microcontroller. The system is developed for use in the field in order to avoid expensive and time-consuming procedures and can be used in a variety of situations to help environmental assessment and control.
Fluidic lenses with variable focal length
Authors: Moran Peter M. , Dharmatilleke Saman , Khaw Aik Hau , Tan Kok Wei , Chan Mei Lin , Rodriguez Isabel
Published: January 2006

We demonstrate in this letter that variable focus lenses can be realized by pressure-induced deformation of a liquid interface. Large deflections can be achieved by exploiting the capillary pressure barrier generated by high surface tension liquids at the exit of a small well. Modulation of the internal pressure of a liquid interface produces a change in its radius of curvature thus a shift in the plane of focus. Using liquids with high surface tension like water and polymeric low surface energy substrates, liquid-air lenses were realized with a wide focal length tunability (optical power for 2 mm lenses from 130 to 350 diopters). Using the liquid-liquid interface of water and a nonmiscible liquid, pressure-induced tunable lenses were also demonstrated. The application of the lenses in imaging was also shown.
Carrier transport and confinement in polarization-induced three-dimensional electron slabs: Importance of alloy scattering in AlGaN
Authors: Simon John , Wang Albert (Kejia) , Xing Huili , Rajan Siddharth , Jena Debdeep
Published: January 2006

By exploiting the difference in spontaneous and piezoelectric polarization between GaN and compositionally graded layers of strained AlGaN, we demonstrate three-dimensional electron slabs of tunable widths (30–100 nm) and densities (1–5×1018cm3). Removal of ionized impurity scattering results in relatively high mobilities limited by alloy scattering at low temperatures, and by a combination of alloy and polar optical phonon scattering at room temperature. Owing to the tunable electron-electron interactions in such slabs, they offer an ideal system to probe high-field transport physics in the III-V nitride semiconductors in general, and the hot-phonon effect in particular.
Buffer-material dependence of InAs quantum dots on GaAs substrate
Authors: Shimizu Hitoshi , Saravanan Shanmugam
Published: January 2006

This letter describes the buffer-material dependence of 1.3-μm-range quantum dots (QDs), a subject that is useful for increasing the dot density while maintaining the photoluminescence (PL) intensity. Four types of buffer material, that is, GaAs, GaAsSb, InGaAs, and Si, were investigated. The PL intensity decreases monotonously with increases in the dot density for all types of buffers. The relationship between the PL intensity and the dot density is almost the same for GaAs, InGaAs, and Si buffer. However, the PL intensity increases about three times when GaAsSb buffer is used. This could be attributed to the reduction of the interfacial defects of the QDs by the surfactant behavior of Sb.
Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents
Authors: Souza Glauco R. , Christianson Dawn R. , Staquicini Fernanda I. , Ozawa Michael G. , Snyder Evan Y. , Sidman Richard L. , Miller J. Houston , Arap Wadih , Pasqualini Renata
Published: January 2006

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au–phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au–phage–imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.
Enigma, a mitochondrial protein affecting lifespan and oxidative stress response in
Drosophila
Authors: Mourikis Philippos , Hurlbut Gregory D. , Artavanis-Tsakonas Spyros
Published: January 2006

Deregulation of energy metabolism by external interventions or mutations in metabolic genes can extend lifespan in a wide range of species. We describe mutations in
Drosophila melanogaster
that confer resistance to oxidative stress and display a longevity phenotype. These phenotypes are associated with molecular lesions in a hitherto uncharacterized gene we named
Enigma.
We show that
Enigma
encodes a mitochondrial protein with homology to enzymes of the β-oxidation of fatty acids and that mutations in this locus affect lipid homeostasis. Our analysis provides further support to the notion that lipid metabolism may play a central role in metazoan lifespan regulation.
Polynomial model approach for resynchronization analysis of cell-cycle gene expression data
Authors: Qiu Peng , Wang Z. Jane , Liu K. J. Ray
Published: January 2006

Motivation: Identification of genes expressed in a cell-cycle-specific periodical manner is of great interest to understand cyclic systems which play a critical role in many biological processes. However, identification of cell-cycle regulated genes by raw microarray gene expression data directly is complicated by the factor of synchronization loss, thus remains a challenging problem. Decomposing the expression measurements and extracting synchronized expression will allow to better represent the single-cell behavior and improve the accuracy in identifying periodically expressed genes.

Results: In this paper, we propose a resynchronization-based algorithm for identifying cell-cycle-related genes. We introduce a synchronization loss model by modeling the gene expression measurements as a superposition of different cell populations growing at different rates. The underlying expression profile is then reconstructed through resynchronization and is further fitted to the measurements in order to identify periodically expressed genes. Results from both simulations and real mircorarray data show that the proposed scheme is promising for identifying cyclic genes and revealing underlying gene expression profiles.

Availability: Contact the authors.

Contact:  qiupeng@umd.edu

Supplementary information: Supplementary data are available at:
The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells
Authors: Regueiro Verónica , Campos Miguel A. , Pons Jaume , Albertí Sebastián , Bengoechea José A.
Published: January 2006

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection withKlebsiella pneumoniae. Infection of A549 and NHBE withK. pneumoniae52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to killK. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-κB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.
Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis
Authors: Burrowes Elizabeth , Baysse Christine , Adams Claire , O'Gara Fergal
Published: January 2006

RsmA is a posttranscriptional regulatory protein in Pseudomonas aeruginosa that works in tandem with a small non-coding regulatory RNA molecule, RsmB (RsmZ), to regulate the expression of several virulence-related genes, including the N-acyl-homoserine lactone synthase genes lasI and rhlI, and the hydrogen cyanide and rhamnolipid biosynthetic operons. Although these targets of direct RsmA regulation have been identified, the full impact of RsmA on cellular activities is not as yet understood. To address this issue the transcriptome profiles of P. aeruginosa PAO1 and an isogenic rsmA mutant were compared. Loss of RsmA altered the expression of genes involved in a variety of pathways and systems important for virulence, including iron acquisition, biosynthesis of the Pseudomonas quinolone signal (PQS), the formation of multidrug efflux pumps, and motility. Not all of these effects can be explained through the established regulatory roles of RsmA. This study thus provides both a first step towards the identification of further genes under RsmA posttranscriptional control in P. aeruginosa and a fuller understanding of the broader impact of RsmA on cellular functions.
Biofilm formation in Campylobacter jejuni
Authors: Joshua G. W. P , Guthrie-Irons C. , Karlyshev A. V. , Wren B. W.
Published: January 2006

The major gastrointestinal pathogen Campylobacter jejuni is shown to exist as three forms of monospecies biofilm in liquid culture. It attaches to a glass surface; forms an unattached aggregate (floc); and forms a pellicle at the liquid–gas interface. The three forms of biofilm resemble each other when examined by scanning electron microscopy. The biofilm mode of growth confers protection against environmental stress, the microaerobic bacteria in flocs surviving up to 24 days at ambient temperature and atmosphere compared to 12 days survival by planktonic bacteria. The wild-type strains C. jejuni 33106, 32799, 33084 and 31485 did not form flocs, and floc formation was reduced in strains mutant in a putative flagellar protein (FliS) and in a phosphate acetyltransferase (Cj0688). All other strains tested, including strains with mutations affecting capsular polysaccharide (kpsM), flagella (maf5), protein glycosylation (pglH) and lipo-oligosaccharide (neuB1) formed flocs. Similarly, all strains tested formed a pellicle and attached to glass except the aflagellate mutant maf5; pellicle formation was reduced in fliS and cj0688 mutants. Different mechanisms, therefore, may control formation of different forms of biofilm. It is proposed that these poorly characterized forms of growth are important for the persistence of C. jejuni in the environment and may in part explain the high incidence of Campylobacter-associated food borne disease.
Homocysteine accumulation causes a defect in purine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs
Authors: Fujita Yasuko , Ukena Eiko , Iefuji Haruyuki , Giga-Hama Yuko , Takegawa Kaoru
Published: January 2006

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of theSchizosaccharomyces pombegenomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designatedmet26. Themet26mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeastSaccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes fromSac. cerevisiaeconstituting the cystathionine pathway (CYS4andCYS3) intoSch. pombeΔmet26cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Δmet26cells was higher than in other methionine auxotrophs and that introduction of theSac. cerevisiaecystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in themet26mutant.
The structural proteome of Pseudomonas aeruginosa bacteriophage ϕKMV
Authors: Lavigne Rob , Noben Jean-Paul , Hertveldt Kirsten , Ceyssens Pieter-Jan , Briers Yves , Dumont Debora , Roucourt Bart , Krylov Victor N. , Mesyanzhinov Vadim V. , Robben Johan , Volckaert Guido
Published: January 2006

The structural proteome of ϕKMV, a lytic bacteriophage infecting Pseudomonas aeruginosa, was analysed using two approaches. In one approach, structural proteins of the phage were fractionated by SDS-PAGE for identification by liquid chromatography-mass spectrometry (LC-MS). In a second approach, a whole-phage shotgun analysis (WSA) was applied. WSA uses trypsin digestion of whole phage particles, followed by reversed-phase HPLC and gas-phase fractionation of the complex peptide mixture prior to MS. The results yield a comprehensive view of structure-related proteins in ϕKMV and suggest subtle structural differences from phage T7.
Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae
Authors: Guiral Sébastien , Hénard Vincent , Laaberki Maria-Halima , Granadel Chantal , Prudhomme Marc , Martin Bernard , Claverys Jean-Pierre
Published: January 2006

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome ofStreptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication inEscherichia coli, but not inS. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, PM, separated from a kanamycin-resistance gene byNcoI andBamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of PMcan be obtained through direct transformation of anS. pneumoniaerecipient with ligation products between that gene andNcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.
The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division
Authors: Dasgupta Arunava , Datta Pratik , Kundu Manikuntala , Basu Joyoti
Published: January 2006

A cluster of genes encoded by ORFs Rv0014c–Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.
3,5-Dihalo-4-hydroxybenzonitriles: isostructures, polymorphs and solvates
Authors: Britton Doyle
Published: January 2006

3,5-Dichloro-4-hydroxybenzonitrile and its solvates with benzene and xylene assemble in chain-like arrangements held together by OH...NC interactions. Two polymorphs each of the corresponding dibromo and diiodo compounds form similar chains which are then bound together by X...X interactions to form approximately planar sheets. These sheets are essentially identical in all four of the bromo and iodo compounds and have approximate p2gg two-dimensional symmetry. These four compounds form an extreme example of two-dimensional isostructuality. The polymorphs differ in the stacking arrangements. One of the Br polymorphs is isostructural with one of the I polymorphs.
Structural disorder of a new zeolite-like lithosilicate, K2.6Li5.4[Li4Si16O38]·4.3H2O
Authors: Park So-Hyun , Boysen Hans , Parise John B.
Published: January 2006

The framework structure of the synthetic microporous lithosilicate RUB-30 (K2.6Li5.4[Li4Si16O38]·4.3H2O) is similar to that of the fibrous zeolites such as natrolite, edingtonite and thomsonite, since all their frameworks include the same secondary structural building unit, the so-called 4–1 T
5O10 cluster of tetrahedra. Unique to the structure of RUB-30, each 4–1 unit consists of a LiSi4O10 moiety within which the single [LiO4] tetrahedron is strictly segregated from the other four [SiO4] tetrahedra. The connection of neighboring 4–1 units through edge-sharing [LiO4] tetrahedra results in a new framework topology. The present work reports an `average' structure of RUB-30 solved by synchrotron X-ray single-crystal diffraction data collected at a second-generation source. A superstructure with a × 2b × c (relative to the subcell quoted above) could be seen in X-ray diffraction data collected with better resolution and higher brightness at a third generation source. Diffuse streaks along k with l = odd and unusual superstructure hkl reflections, with k = odd and l = odd only, indicate a more complicated real structure of the material. To explain this observation we propose two different structure types which are statistically, but coherently, intergrown in RUB-30.
Systematic prediction of new ferroelectrics in space group R3. I
Authors: Abrahams S. C.
Published: January 2006

Release 2005/1 of the Inorganic Crystal Structure Database contains 158 entries under the space group R3. The first 81 together with 15 related entries include 52 different structure types, 10 of which represent families that have two or more isostructural members. There are 18 types that satisfy the structural criteria for ferroelectricity with a confidence level that necessarily depends upon the reliability of each structural determination. Among them, (LiTaO3)9·Ta2O5 and the CsCd(NO2) family are known ferroelectrics. The remaining 16 include the high-intensity higher-order harmonic generator II-BaB2O4, not previously recognized as ferroelectric. The other predicted ferroelectrics are Te(OH)6·NaF, the MgSO3·6H2O family, Li3P3O9·3H2O, SeNbF9, Al2(H2PO4)3PO4·6H2O, [Cr(OCN2H4)6]·[(Co(NH3)2(NO2)4]3·(H2O)1.5], NaZnBr3·5H2O, III-Ag3SI, Tl2S, the Ba3Yb4O9 family, the Fe10.7Ru1.3As5 family, Sr17Ta10S42, Pb8Sb3 and the Sc7I12C family. Six structures reported in the space group R3 most likely exhibit mirror symmetry, with three experimental confirmations. The other 19 structures are most likely nonpolar, with six experimental confirmations. The capacity for physical property prediction by the remaining ten structures is shown to be in doubt. Thus, 46% of the present 52 inorganic structure types initially determined in the space group R3 and 35% of those currently assigned to R3 more likely exhibit an additional inversion center, mirror or glide plane, strongly exceeding the recently reported error rate of ∼ 11% in organic structures.
Variation of loop sequence alters stability of cytolethal distending toxin (CDT): Crystal structure of CDT from Actinobacillus actinomycetemcomitans
Authors: Yamada Taro , Komoto Junichi , Saiki Keitarou , Konishi Kiyoshi , Takusagawa Fusao
Published: January 2006

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 Å resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic “aromatic patch” on CdtA.
Crystal structural analysis and metal‐dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+
Authors: Doudeva Lyudmila G. , Huang Hsinchin , Hsia Kuo‐Chiang , Shi Zhonghao , Li Chia‐Lung , Shen Yongliang , Cheng Yi‐Sheng , Yuan Hanna S.
Published: January 2006

The nuclease domain of ColE7 (N‐ColE7) contains an H‐N‐H motif that folds in a ββα‐metal topology. Here we report the crystal structures of a Zn2+‐bound N‐ColE7 (H545E mutant) in complex with a 12‐bp duplex DNA and a Ni2+‐bound N‐ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 Å and 2.0 Å, respectively. Metal‐dependent cleavage assays showed that N‐ColE7 cleaves double‐stranded DNA with a single metal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ atconcentrations of <25mM, indicating thatzincisthe physiologically relevant metal ion in N‐ColE7 in host E. coli. In the crystal structure of N‐ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni2+ is bound in N‐ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His‐metal finger motif can be coordinated to six ligands, such as Mg2+ in I‐PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ in ColE7. Universally, the metal ion in the His‐metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P–O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.
1α,25‐Dihydroxyvitamin D3 downmodulates the functional differentiation of Th1 cytokine‐conditioned bone marrow‐derived dendritic cells beneficial for cytotoxic T lymphocyte generation
Authors: Matsuzaki Junko , Tsuji Takemasa , Zhang Yue , Wakita Daiko , Imazeki Ikuo , Sakai Tomoaki , Ikeda Hiroaki , Nishimura Takashi
Published: January 2006

Various dendritic cell subsets are induced from bone marrow cells under different cytokine conditions. We have demonstrated previously that the Th1‐cytokine‐conditioned bone marrow‐derived dendritic cell (BMDC) subset BMDC1 (generated in the presence of granulocyte‐macrophage colony‐stimulating factor [GM‐CSF] + interleukin [IL]‐3 + interferon [IFN]‐γ+ IL‐12) induces a much stronger type 1 immune response than BMDC0 (GM‐CSF + IL‐3). In the present study, we investigated the effect of 1α,25‐dihydroxyvitamine D3 (VitD3), which is a known immunomodulating drug, on the differentiation of BMDC subsets. The addition of VitD3 significantly influenced the functional differentiation of BMDC1 compared with BMDC0. Specifically, the addition of VitD3 greatly decreased the expression levels of MHC class I, CD80, CD40 and leukocyte function‐associated antigen (LFA)‐1 molecules on BMDC1. In addition, VitD3‐treated BMDC1 (VD3‐BMDC1) almost completely lost their immunostimulating activity for inducing type 1 immunity and cytotoxic T lymphocyte generation. A failure in the induction of type 1 immunity by VD3‐BMDC1 appeared to be due to the following: (i) the expression of co‐stimulatory molecules on VD3‐BMDC1 was strongly downmodulated compared with BMDC1 generated without VitD3; and (ii) VD3‐BMDC1 showed significantly lower mRNA expression of IFN‐γ and IFN‐β, factors that are essential for cytotoxic T lymphocyte induction. VitD3 inhibited the differentiation of functionally competent BMDC1 during the early phase of differentiation but not during the late differentiation period. A possible reason for the inhibition of BMDC1 differentiation by VitD3 is reduced phosphorylation of STAT1 during early differentiation. Taken together, VitD3 strongly suppressed T‐cell responses by inhibiting functional differentiation of precursor dendritic cells into functional BMDC1 that are feasible for inducing Th1‐dependent cellular immunity. (Cancer Sci 2006; 97: 139 –147)