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PubMed1992

Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.

Amino Acid Sequence↗
PubMed2004

Meta-analysis of clinical characteristics of 299 carriers of LMNA gene mutations: do lamin A/C mutations portend a high risk of sudden death?

This study evaluated common clinical characteristics of patients with lamin A/C gene mutations that cause either isolated dilated cardiomyopathy or dilated cardiomyopathy in association with skeletal muscular dystrophy. We pooled clinical data of all published carriers of lamin A/C gene mutations as cause of skeletal and/or cardiac muscle disease and reviewed ECG findings. Cardiac dysrhythmias were reported in 92% of patients after the age of 30 years; heart failure was reported in 64% after the age of 50. Sudden death was the most frequently reported mode of death (46%) in both the cardiac and the neuromuscular phenotype. Carriers of lamin A/C gene mutations often received a pacemaker (28%). However, this intervention did not alter the rate of sudden death. Review of the ECG findings typically showed a low amplitude P wave and prolongation of the PR interval with a narrow QRS complex. This meta-analysis suggests that cardiomyopathy due to lamin A/C gene mutations portends a high risk of sudden death, and that this risk does not differ between subjects with predominantly cardiac or neuromuscular disease. This implies then that all carriers of a lamin A/C gene mutation need to be carefully screened with particular emphasis also on tachyarrhythmias. Prospective studies are needed to evaluate risk stratification and proper treatment strategies.

Arrhythmias, Cardiac↗
PubMed2005

Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization.

Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.

Amino Acid Sequence↗
PubMed1996

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.

Base Sequence↗
PubMed2006

Hyperglycemia stimulates coagulation, whereas hyperinsulinemia impairs fibrinolysis in healthy humans.

Type 2 diabetes and insulin resistance syndromes are associated with an increased risk for cardiovascular and thrombotic complications. A disturbed balance between coagulation and fibrinolysis has been implicated in the pathogenesis hereof. To determine the selective effects of hyperglycemia and hyperinsulinemia on coagulation and fibrinolysis, six healthy humans were studied on four occasions for 6 h: 1) lower insulinemic-euglycemic clamp, 2) lower insulinemic-hyperglycemic clamp, 3) hyperinsulinemic-euglycemic clamp, and 4) hyperinsulinemic-hyperglycemic clamp. In the hyperglycemic clamps, target levels of plasma glucose were 12 versus 5 mmol/l in the normoglycemic clamps. In the hyperinsulinemic clamps, target plasma insulin levels were 400 versus 100 pmol/l in the lower insulinemic clamps. Hyperglycemia exerted a procoagulant effect irrespective of insulin levels, as reflected by mean twofold rises in thrombin-antithrombin complexes and soluble tissue factor, whereas hyperinsulinemia inhibited fibrinolysis irrespective of glucose levels, as reflected by a decrease in plasminogen activator activity levels due to a mean 2.5-fold rise in plasminogen activator inhibitor type 1. The differential effects of hyperglycemia and hyperinsulinemia suggest that patients with hyperglycemia due to insulin resistance are especially susceptible to thrombotic events by a concurrent insulin-driven impairment of fibrinolysis and a glucose-driven activation of coagulation.

Adult↗
PubMed2003

Mechanisms for nucleosome mobilization.

Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics.

DNA↗
PubMed2004

SATB1 family protein expressed during early erythroid differentiation modifies globin gene expression.

Special AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with epsilon-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases gamma-globin and down-regulates SATB1 family protein and epsilon-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the beta-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the epsilon-globin promoter. SATB1 overexpression increases epsilon-globin and decreases gamma-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the epsilon-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the gamma-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in epsilon-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the epsilon-globin gene by interaction with specific sites in the beta-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation.

Cell Differentiation↗
PubMed2006

Public health preparedness in Alberta: a systems-level study.

BACKGROUND: Recent international and national events have brought critical attention to the Canadian public health system and how prepared the system is to respond to various types of contemporary public health threats. This article describes the study design and methods being used to conduct a systems-level analysis of public health preparedness in the province of Alberta, Canada. The project is being funded under the Health Research Fund, Alberta Heritage Foundation for Medical Research. METHODS/DESIGN: We use an embedded, multiple-case study design, integrating qualitative and quantitative methods to measure empirically the degree of inter-organizational coordination existing among public health agencies in Alberta, Canada. We situate our measures of inter-organizational network ties within a systems-level framework to assess the relative influence of inter-organizational ties, individual organizational attributes, and institutional environmental features on public health preparedness. The relative contribution of each component is examined for two potential public health threats: pandemic influenza and West Nile virus. DISCUSSION: The organizational dimensions of public health preparedness depend on a complex mix of individual organizational characteristics, inter-agency relationships, and institutional environmental factors. Our study is designed to discriminate among these different system components and assess the independent influence of each on the other, as well as the overall level of public health preparedness in Alberta. While all agree that competent organizations and functioning networks are important components of public health preparedness, this study is one of the first to use formal network analysis to study the role of inter-agency networks in the development of prepared public health systems.

Alberta↗
PubMed2006

PKR1 encodes an assembly factor for the yeast V-type ATPase.

Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Delta cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector.

Amino Acid Sequence↗
PubMed2006

The clinical nursing competences and their complexity in Belgian general hospitals.

AIM: This paper reports a study whose aim was to chart clinical nursing competences and their complexity in Belgian general hospitals. BACKGROUND: Competence is an essential factor for assuring quality, safety and cost-effective health care. As clinical competence cannot be evaluated separately from the clinical context in which decisions are made, competence is defined as functional adequacy and the capacity to integrate knowledge and skills with attitudes and values into the specific contexts of practice. METHOD: The study took place in 2003 and focused on basic care, specialized care and elder care. Head nurses of 176 departments processed 521 patient situations. Experts selected 50 situations per area. In a Delphi procedure 100 experts described the nursing competences required in each situation necessary to provide quality acceptable care. The experts determined the global complexity of each of these competences as well as the complexity of the cognitive, psychomotor and affective aspects. FINDINGS: The global complexity of the various care areas was similar. Cognitive and affective aspects of competences scored higher than psychomotor aspects. Simple and average situations did not show any significant difference with regard to complexity and were less complex than difficult situations, which obtained the highest complexity score. The complexity of emergency situations did not differ from that of difficult situations. CONCLUSION: Emergency and difficult situations require more competences than offered by basic nursing education. The continuous presence of care providers with additional education is necessary for each care area to respond adequately to emergency and difficult situations. Nurses with additional academic qualifications need to be available for consultation. There are only a few possibilities of employing lower qualified staff in direct care, with the exception of support with regard to administrative tasks. Cognitive and affective aspects of the competences require particular attention during the education programme.

Adult↗
PubMed2006

Macrophage reprogramming by mycolic acid promotes a tolerogenic response in experimental asthma.

RATIONALE: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function. OBJECTIVES: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity. METHODS: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function. MEASUREMENTS AND MAIN RESULTS: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages. CONCLUSIONS: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Animals↗
PubMed2003

Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides.

Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that oxidation of some methionine residues occurred to a minor extent during the solid-phase isolation steps.

Humans↗
PubMed2006

Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate.

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.

Amino Acid Sequence↗
PubMed2002

Characterization of Pseudoalteromonas citrea and P. nigrifaciens isolated from different ecological habitats based on REP-PCR genomic fingerprints.

DNA primers corresponding to conserved repetitive interspersed genomic motifs and PCR were used to show that REP, ERIC and BOX-like DNA sequences are present in marine, oxidative, gram-negative Pseudoalteromonas strains. REP, ERIC and BOX-PCR were used for rapid molecular characterization of both the type species of the genus and environmental strains isolated from samples collected in different geographical areas. PCR-generated genomic fingerprint patterns were found to be both complex and strain specific. Analysis of the genotypic structure of phenotypically diverse P. citrea revealed a geographic clustering of Far Eastern brown-pigmented, agar-digesting strains of this species. Marine isolates of P. nigrifaciens with 67-70% DNA relatedness generated genomic patterns different from those of the type strain and formed a separate cluster. It is concluded that REP, ERIC and BOX-PCR are effective in generating strain specific patterns that can be used to elucidate geographic distribution, with these genomic patterns providing a valuable biogeographic criterion.

DNA Fingerprinting↗
PubMed2007

Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors.

Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.

Animals↗
PubMed2003

Structure of pteridine reductase (PTR1) from Leishmania tarentolae.

The protozoan parasites Leishmania utilize a pteridine-reducing enzyme, pteridine reductase (PTR1), to bypass antifolate inhibition. The crystal structure of PTR1 from L. tarentolae has been solved as a binary complex with NADPH at 2.8 A resolution. The structure was solved by molecular-replacement techniques using the recently reported L. major PTR1 structure as a search model. Comparisons of the present structure with the L. major PTR1 allowed us to identify regions of flexibility in the molecule. PTR1 is a member of the growing family of short-chain dehydrogenases (SDR) which share the characteristic Tyr(Xaa)(3)Lys motif in the vicinity of the active site. The functional enzyme is a tetramer and the crystallographic asymmetric unit contains a tetramer with 222 point-group symmetry.

Amino Acid Sequence↗
PubMed1994

Characteristics of visual interference with visuospatial working memory.

Recent discussions of visuospatial working memory have suggested that this subsystem may incorporate a visual buffer which holds visuospatial information relatively passively. Empirical investigations of visual interference with information held within a visuospatial subsystem have yielded somewhat equivocal results. Nonetheless, evidence from Logie (1986) has indicated that visuospatial processing can be disrupted by passive exposure to irrelevant visual material in a manner analogous to the disruption of serial verbal recall by exposure to irrelevant speech. This paper reports two experiments which explore whether such irrelevant visual input is disruptive to storage of imaginal information in a primarily spatial task--the Brooks spatial matrix task. Experiment 1 shows that exposure to irrelevant visual input during encoding selectively disrupts performance on a spatial, but not a verbal, version of the task. The extent of such disruption is shown to be independent of the visual complexity of the material, its similarity to the to-be-remembered information, or a change in state, with a static white square pattern yielding equivalent disruption to that produced by changing matrix patterns. The second experiment indicates that this pattern of effects is robust, and that such disruption is evident at an equivalent level when the visual material is present only during a 20-second retention interval. These results are interpreted as evidence of obligatory access of external visual material to a passive visual buffer. Implications for the nature of a visuospatial subsystem in working memory are discussed.

Adult↗
PubMed2007

Free energy profiles for monomer capture in Grubbs- and SHOP-type olefin polymerization catalysts: a constraint ab initio molecular dynamics study.

Density functional theory together with Car-Parrinello ab initio molecular dynamics simulation has been used to investigate the free energy profiles (FEP) of monomer capture in Grubbs- and SHOP-type olefin polymerization catalysts. The FEPs along the reaction coordinates at 300 K were determined directly by a point wise thermodynamic integration technique. Comparison between potential energy profile (PEP) and the FEP has been made. The results show that, for both catalysts, the PEP for the monomer ethylene uptake by the metal center is a typical Morse curve without energy barrier. However, a small barrier (1.8 kcal/mol for Grubbs catalyst and 2.4 kcal/mol for SHOP catalyst) exists on the FEP. The pi complexation energy on the FES at 300 K is higher by 10-12 kcal/mol over that on the PES. The differences between FES and PES are due to entropy contribution. Slow growth simulations on the ethylene capture process show that the ethylene attacks the metal center by an asynchronous mode. This indicates that the forming of the pi-bonding between the metal and ethylene is initiated by electrophilic attack of the metal to one of the ethylene carbons.

Alkenes↗