Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.

Use of adhesion counts to help predict symptomatic infection and the ability of fluoroquinolones to penetrate bacterial biofilms on the bladder cells of spinal cord injured patients

There were three objectives to the present study: (1) compare the bladder infection rate and extent of biofilm formation for seven untreated spinal cord injured (SCI) patients and seven given prophylactic co-trimoxazole, (2) identify a level of bacterial adhesion to bladder cells which could be used to help predict symptomatic infection, and (3) determine from in vivo and in vitro studies whether fluoroquinolones were effective at penetrating bacterial biofilms. The results showed that the infection rate had not changed with the introduction of prophylaxis. However, the uropathogenic population had altered subsequent to the introduction of prophylaxis with E. coli being replaced by E. faecalis as the most common cause of infection. In 63% of the specimens from asymptomatic patients, the bacterial counts per cell were <20, while 81% of specimens from patients with at least one sign and one symptom of urinary tract infection (UTI) had > 20 adherent bacteria per bladder cell. Therefore, it is proposed that counts of > 20 bacteria adherent to sediment transitional epithelial bladder cells may be predictive of symptomatic UTI. Clinical data showed that fluoroquinolone therapy reduced the adhesion counts to <20 per cell in 63% of cases, while trimethoprim-sulfamethoxazole only did so in 44%. Further in vitro testing showed that ciprofloxacin (0.1, 0.5 and 1.0 micrograms/ml) partially or completely eradicated adherent biofilms from 92% of spinal cord injured patients' bladder cells, while ofloxacin did so in 71% cases and norfloxacin in 56%. These findings have important implications for the detection and treatment of bacteriuria in spinal cord injured patients.

The role of ECE1 variants in cognitive ability in old age and Alzheimer's disease risk

The ß-amyloid peptide may play a central role in Alzheimer's disease (AD) pathogenesis. We have evaluated variants in seven Aß-degrading genes (ACE, ECE1, ECE2, IDE, MME, PLAU, and TF) for association with AD risk in the Genetic and Environmental Risk in Alzheimer's Disease Consortium 1 (GERAD1) cohort, and with three cognitive phenotypes in the Lothian Birth Cohort 1936 (LBC1936), using 128 and 121 SNPs, respectively. In GERAD1, we identified a significant association between a four-SNP intragenic ECE1 haplotype and risk of AD in individuals that carried at least one APOE e4 allele (P = 0.00035, odds ratio = 1.61). In LBC1936, we identified a significant association between a different two-SNP ECE1 intragenic haplotype and non-verbal reasoning in individuals lacking the APOE e4 allele (P = 0.00036, ß = -0.19). Both results showed a trend towards significance after permutation (0.05 <P <0.10). A follow-up cognitive genetic study evaluated the association of ECE1 SNPs in three additional cohorts of non-demented older people. Meta-analysis of the four cohorts identified the significant association (Z <0.05) of SNPs in the ECE-1b promoter with non-verbal reasoning scores, particularly in individuals lacking the APOE e4 allele. Our genetic findings are not wholly consistent. Nonetheless, the AD associated intronic haplotype is linked to the 338A variant of known ECE1b promoter variant, 338C>A (rs213045). We observed significantly less expression from the 338A variant in two human neuroblastoma cell lines and speculate that this promoter may be subject to tissue-specific regulation.

PROBLEM-SOLVING IN SCHIZOPHRENIA - A SPECIFIC DEFICIT IN PLANNING ABILITY

The study investigated problem solving ability in schizophrenia. Thirty DSM-IIIR schizophrenic patients and 27 matched normal controls were tested on the Three-Dimensional Computerised Tower of London Test (3-D CTL Test) (Morris et al., 1993). The patients took significantly more moves to solve a series of problems and solved significantly fewer problems in the predetermined minimum number of moves. The patients response times, as measured using a control version of the task (the 3-D CTL Control), were longer than those of the controls. However, when latencies were adjusted to take into account the slower responses overall, the patients planning times were not significantly increased. Inaccurate planning, as defined by taking more moves, did not correlate with either positive or negative symptoms, but the response times tended to be longer in patients who had more negative symptoms. The findings suggest that there is a deficit in problem solving activity in schizophrenia that may be associated with translating 'willed intentions' into action, independent of slower motor speed.

The link between logic, mathematics and imagination: evidence from children with developmental dyscalculia and mathematically gifted children

This study examined performance on transitive inference problems in children with developmental dyscalculia (DD), typically developing controls matched on IQ, working memory and reading skills, and in children with outstanding mathematical abilities. Whereas mainstream approaches currently consider DD as a domain-specific deficit, we hypothesized that the development of mathematical skills is closely related to the development of logical abilities, a domain-general skill. In particular, we expected a close link between mathematical skills and the ability to reason independently of one's beliefs. Our results showed that this was indeed the case, with children with DD performing more poorly than controls, and high maths ability children showing outstanding skills in logical reasoning about belief-laden problems. Nevertheless, all groups performed poorly on structurally equivalent problems with belief-neutral content. This is in line with suggestions that abstract reasoning skills (i.e. the ability to reason about content without real-life referents) develops later than the ability to reason about belief-inconsistent fantasy content.A video abstract of this article can be viewed at http://www.youtube.com/watch?v=90DWY3O4xx8.

The Houses of the Oireachtas:Parliament in Ireland

The Irish parliament - the Oireachtas - is nearing the centenary year of its foundation, making it one of the oldest continuously surviving parliaments in the world. As the most important national institution in the state, it plays an essential role in giving voice to a diversity of views and opinions, providing stable governments, approving law and national budgets and upholding democratic values. For much of its existence, however, and most pointedly in the context of recent banking and economic crises, it has been subject to criticism concerning its ability to adequately hold the executive to account, to act as a coherent policy-making forum, to meet the challenges arising from European Union membership, to embrace wide-ranging reforms and to develop with purpose and ambition.

This comprehensive new volume considers all aspects of the Houses of the Oireachtas - including their evolution, composition, organisation, financing, administration and reform. Contributors include academics, administrators and sitting and former parliamentarians. Contemporary challenges brought about by transformations in media style, increased inter-parliamentarism and the changing character of politics are also addressed. The book questions a number of assumptions about parliament and its work, including the efficacy of the legislative and budgetary processes, the nature of executive-legislative relations and the perceived encroachment of the courts on the legislature. Combined, this wide-ranging and detailed study fills a long-standing void, and provides essential reading not alone for those interested in Irish politics and government, but also for students and scholars of legislative studies.

A longitudinal analysis of estimation, counting skills, and mathematical ability across the first school year

In response to claims that the quality (and in particular linearity) of children's mental representation of number acts as a constraint on number development, we carried out a longitudinal assessment of the relationships between number line estimation, counting, and mathematical abilities. Ninety-nine 5-year-olds were tested on 4 occasions at 3 monthly intervals. Correlations between the 3 types of ability were evident, but while the quality of children's estimations changed over time and performance on the mathematical tasks improved over the same period, changes in one were not associated with changes in the other. In contrast to the earlier claims that the linearity of number representation is potentially a unique contributor to children's mathematical development, the data suggest that this variable is not significantly privileged in its impact over and above simple procedural number skills. We propose that both early arithmetic success and estimating skill are bound closely to developments in counting ability.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells

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 with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, 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 kill K. 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-kappaB. 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.

Comparison of diagnostic ability between a fast strategy, tendency-oriented perimetry, and the standard bracketing strategy

Purpose: To compare the diagnostic abilities of the standard bracketing strategy (BR) and a fast strategy, the tendency-oriented perimetry (TOP). Methods: Seventy-seven controls and 91 eyes from patients with glaucoma were analyzed with the strategies TOP and BR. Sensitivity (Se), specificity (Sp), the area under the receiver operating characteristic (ROC) curve (AC) and the optimum cutoff value (CO) were calculated for the visual field indices mean defect (MD), the square root of the loss variance (sLV) and the number of pathological points (NPP). Results: In the glaucoma group, the mean MD value using TOP and BR was 7.5 and 8.3 dB, respectively. The mean sLV value using TOP and BR was 5.0 and 5.3 dB, respectively. Indices provided by TOP had higher ROC values than the ones provided by BR. Using TOP, the index with the best diagnostic ability was sLV (Sp = 94.8, Se = 90.1, AC = 0.966, CO = 2.5 dB), followed by NPP and MD. Using BR, the best results were obtained for MD (Sp = 92.2, Se = 81.3, AC = 0.900, CO = 2.5 dB) followed by sLV and NPP. Conclusions: A fast strategy, TOP, had superior diagnostic ability than the standard BR. Although TOP provided lower LV values than BR, the diagnostic ability of this index was higher than that of the conventional strategy. Copyright © 2005 S. Karger AG.

Peer tutoring in reading: The effects of role and organization on two dimensions of self-esteem

Background. Paired reading (PR) is an application of peer tutoring. It has been extensively researched, and its efficacy across a range of outcomes has been established. Benefits include improvements in key reading skills, and also in affective aspects of learning. Several studies have shown gains in self-esteem, although measurement methods have varied, and the model of self-esteem has rarely been clearly articulated.
Aims. To investigate the changes in self-esteem of children participating in a randomized trial of PR over a 15-week treatment period. To investigate the relative contribution of self-worth and self-competence to any gains in self-esteem. To investigate whether the pattern of change differs in children who take on different roles in the PR process.
Participants. The participants comprised a subset of a large-scale randomized trial of peer learning (The Fife Peer Learning Project). Four schools were randomly selected from schools allocated to the same-age PR condition, and four schools from those allocated to the cross-age PR condition. The same-age group consisted of 87 primary 6 children (10–11 years old). The cross-age group consisted of 81 primary 6 children. The controls, from schools randomly selected from a neighbouring authority, consisted of 92 primary 6 children.
Method. A pre–post design employing self-report measures of self-esteem. Rosenberg’s Self-Esteem Scale was used, with scores analysed for worth and competence. The treatment period was 15 weeks, with the participants following a prescribed PR process.
Results. Significant pre–post gains were noted in self-esteem, driven predominantly by improved beliefs about competence, in both same-age and cross-age conditions, but not for controls. Gains were also seen in self-worth in the cross-age condition. Further analyses of the influence of organizational condition (same-age or cross-age) and role played (tutor vs. tutee) showed significant differences between same-age tutors and cross-age tutors in relation to self-worth. Effect sizes were generally small or moderate.
Conclusions. The findings provide further support for the belief that PR can enhance self-esteem. Importantly, the use of a two-dimensional model provides extra information about self-perceptions in PR contexts: first, the central role of self- competence; and second, the gains in self-worth which are associated with tutoring younger children (but not same-age peers). This new information has educational significance for schools considering the potential of peer tutoring and the benefits of different organizational conditions.