bluuebunny/crossref_metadata_2025 huggingface dataset:
Feeding the immune system: the role of micronutrients in restoring resistance to infections.
Authors: Maggini S. , Beveridge S. , Sorbara P. J. P. , Senatore G.
Published: February 2009

The immune system protects the body against pathogens and cancer cells, thereby defending it against infections and diseases via three main components: epithelial barriers, immune cells and proteins including antibodies. Immune defences are constantly active and consist of a complex network of coordinated biological responses. Therefore, the immune system needs to be fed properly with energy sources and essential micronutrients serving as cofactors in the development, maintenance and expression of the immune response. The general nutritional status of an individual modulates immune functions, and immunocompetence is regarded as a measure of adequate nutrition. Low levels of vitamins, minerals and trace elements lead to suppressed immunity, predisposing individuals to infections, which in turn aggravate the nutritional status, leading to a vicious cycle. Supplying the deficient micronutrients with the diet or a dietary supplement can re-establish immune function. A sufficient and balanced diet should cover the overall micronutrient requirements. However, many population segments both in developing and industrialized countries do not get adequate amounts of essential micronutrients through the diet. Current environmental and geopolitical developments (i.e. global economic crisis, rice crisis and other food shortages, the increase in maize cultivation for ethanol production, etc.) are exacerbating problems of nutritional status in many parts of the world and represent a serious threat to nutritional health. This review focuses on nutritional aspects of the immune system and discusses the roles of vitamins A, D, E, C, B
6
, B
12
and folate as well as of the trace elements selenium, zinc, iron and copper in supporting the body's natural defence system and restoring resistance to infections by enhancing the three levels of immunity: epithelial barriers, immune cells and antibody production.
The Sexual Economy of the Islamic Republic
Authors: Afary Janet
Published: February 2009

This article examines the gender and sexual policies of the Islamic Republic and their ramifications. It argues that the policies of the Islamist government cannot easily be categorized as “puritanical” or “moralistic.” Rather we can argue that various factions within the state actively deployed a new “sexual economy” for the population. Sometimes, the Islamist state privileged patriarchal interpretations of gender norms over more modern ones. At other times, it adopted modern projects such as family planning alongside a discourse that presented them as practices rooted in traditional Islam. In all cases, the state used modern institutions to disseminate and enforce these practices.
Undercounting Women's Work in Iran
Authors: Moghadam Fatemeh Etemad
Published: February 2009

This paper explores the possibility of a considerable undercounting of women's labor force participation in the official surveys in Iran. According to the official data, the proportion of females in the total active labor force was about 15.5 percent in 2006. Furthermore, according to the official data the share of female in total active labor in agriculture was about 10 percent in 1996. An examination of a large body of field research on the subject, however, suggests a much higher participation rate, about 40 percent of total agricultural labor. This paper will examine these studies and explore the reasons behind the underestimations. Pointing at the growing visibility of urban women in public space, the increasing share of skilled and educated women, rising cost of living, and the need for both male and female incomes to support an urban family, some observers have suggested that the official data underestimate urban female labor participation, as well. Informal urban labor, however, has not been adequately studied. Anecdotal information, however, suggests the existence of a significant female informal economy in both traditional and modern industries. In 2001, I undertook a micro study of 350 working age women in the affluent northern part of Tehran and found that a large number of educated upper and middle class women were active in the informal market. This finding was in sharp contrast to the studies in other developing countries in which informal participants are generally poor and unskilled and are unable to join the modern formal economy. This paper will also explore the reasons for undercounting of urban female labor.
The Case of Mohammad Khordadian, an Iranian Male Dancer
Authors: Papan-Matin Firoozeh
Published: February 2009

In his speech at Columbia University, President Mahmud Ahmadinejad stated that there are no homosexuals in Iran. His attempt was to avoid the subject and any ensuing controversies that could hamper the nuclear talks at the United Nations General Assembly the following day. The present study will argue that the denial of homosexuality has been selectively applied to situations when the most prudent alternative was to overlook the issue or to keep quiet about it. A few years earlier, while the former president, Mohammad Khatami, was still in office, this attitude determined the course of action in dealing with Mohammad Khordadian, a famous Los Angeles based Iranian-American male cabaret dancer who was also rumored to be gay. In the spring of 2002, Khordadian visited Iran for the first time in more than twenty years. The authorities considered it an offense that he dared to challenge the morality codes of the Islamic Republic by crossing the border into Iran without any apprehension. The imprisonment and trial of Khordadian is a unique case in focus.
The determinants of undergraduate degree performance: How important is gender?
Authors: Barrow Michael , Reilly Barry , Woodfield Ruth
Published: February 2009

This study uses data drawn from three recent cohorts of undergraduates at the University of Sussex to investigate the key determinants of degree performance. The primary theme of the study is an examination of the gender dimension to degree performance. The average ‘good’ degree rate for female students was found to be superior to the male rate. The modest raw gender differential in first class degree rates favoured women but was found to be attributable to their better endowments, particularly pre‐entry qualifications. The largest differential favouring women was in the II:i classification, where almost all of the difference was attributable to differentials in coefficient treatment rather than endowments (or characteristics). The analysis undertaken also allowed the investigation of a number of sub‐themes relating to the effects on degree performance of, inter alia, pre‐entry qualifications, ethnicity, socio‐economic background and health disability. The largest effects were reserved for the role of pre‐entry qualifications with more modest effects detected for ethnicity and socio‐economic background.
‘This baby…it isn't alive.’ Towards a community of learners for vocational orientation
Authors: Boersma Annoesjka , ten Dam Geert , Volman Monique , Wardekker Wim
Published: February 2009

The aim of this study is to contribute to the innovation of pre‐vocational education, in particular, students' orientation at possible future occupations. From our theoretical understanding, vocational orientation that makes sense to students requires them to be part of a ‘community of learners for vocational orientation’. In such a community, students are stimulated to learn deliberately while participating in vocational practices. In this article we distinguish four parameters to define a community of learners for vocational orientation. We then present the results of a collective instrumental case study into teaching‐learning processes in four classes in two pre‐vocational secondary schools. Data were collected through classroom observations, a series of interviews with teachers and students. The interviews focused on eight ‘critical incidents’ recorded during classroom observations. The results show that the parameters ‘shared learning’ and ‘meaningful learning’ are more manifest in the teaching‐learning process than ‘reflective learning’ and ‘a focus on transferable learning outcomes’. The article concludes with a discussion of the limits of working in simulated work situations in school for realizing effective pre‐vocational education.
Moving beyond the metaphor of transfer of learning
Authors: Hager Paul , Hodkinson Phil
Published: February 2009

This paper argues that much contemporary educational policy makes assumptions about learning that are directly contradicted by the best research and theorising of learning that has occurred over the last decade and more. This worrying mismatch is largely attributable to adherence by policy makers (and other key stakeholders such as employers), to ‘common sense’ notions of learning transfer. In fact, these ‘common sense’ notions of transfer have increasingly been discarded even in the learning transfer literature. However, we go further in arguing that transfer is a totally inappropriate metaphor for thinking about most learning, but especially for vocational learning. Accepting that thought about learning inevitably involves metaphors, we consider the merits and otherwise of various other learning metaphors including participation and construction. We conclude that the conceptual flaws of transfer can be avoided by employing alternative metaphors. The value of our recommended alternative is illustrated by its power to illuminate data on learning collected from various research projects.
Gender and Eloquence in Ercole de' Roberti'sPortia and Brutus
Authors: Cox Virginia
Published: February 2009

A commonplace of modern feminist scholarship holds that fifteenth-century Italian humanists regarded the figure of the articulate women with hostility and suspicion. This position is insufficiently nuanced: while it may have been true to some extent in republican contexts, it was emphatically not the case in the secular princely courts, where women's capacity for eloquence was frequently a subject of praise. Humanistic attitudes toward female eloquence are examined here with special reference to Ercole de’ Roberti's representation of the classical heroine Portia in oratorical guise in his Portia and Brutus, painted at the court of Ferrara in the late 1480s or early ’90s. The article contextualizes Roberti's painting with regard to its classical literary sources, to contemporary practices of female oratory, and to the cultural and social self-positioning of the work's probable patron, Duchess Eleonora d'Aragona.
“The Root is Hidden and the Material Uncertain”: The Challenges of Prosecuting Witchcraft in Early Modern Venice*
Authors: Seitz Jonathan
Published: February 2009

The rich archival records of the Holy Office of the Inquisition in Venice have yielded much information about early modern society and culture. The transcripts of witchcraft trials held before the Inquisition reveal the complexities of early modern conceptions of natural and supernatural. The tribunal found itself entirely unable to convict individuals charged with performing harmful magic, or maleficio, as different worldviews clashed in the courtroom. Physicians, exorcists, and inquisitors all had different approaches to distinguishing natural phenomena from supernatural, and without a consensus guilty verdicts could not be obtained.
Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation
Authors: Schafer Blanca , Quispe Joel , Choudhary Vineet , Chipuk Jerry E. , Ajero Teddy G. , Du Han , Schneiter Roger , Kuwana Tomomi
Published: February 2009

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.
Syndapin Promotes Formation of a Postsynaptic Membrane System in Drosophila
Authors: Kumar Vimlesh , Fricke Robert , Bhar Debjani , Reddy-Alla Suneel , Krishnan K. S. , Bogdan Sven , Ramaswami Mani
Published: February 2009

Syndapins belong to the F-BAR domain protein family whose predicted functions in membrane tubulation remain poorly studied in vivo. At Drosophila neuromuscular junctions, syndapin is associated predominantly with a tubulolamellar postsynaptic membrane system known as the subsynaptic reticulum (SSR). We show that syndapin overexpression greatly expands this postsynaptic membrane system. Syndapin can expand the SSR in the absence of dPAK and Dlg, two known regulators of SSR development. Syndapin's N-terminal F-BAR domain, required for membrane tubulation in cultured cells, is required for SSR expansion. Consistent with a model in which syndapin acts directly on postsynaptic membrane, SSR expansion requires conserved residues essential for membrane binding in vitro. However, syndapin's Src homology (SH) 3 domain, which negatively regulates membrane tubulation in cultured cells, is required for synaptic targeting and strong SSR induction. Our observations advance knowledge of syndapin protein function by 1) demonstrating the in vivo relevance of membrane remodeling mechanisms suggested by previous in vitro and structural analyses, 2) showing that SH3 domains are necessary for membrane expansion observed in vivo, and 3) confirming that F-BAR proteins control complex membrane structures.
Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast
Authors: Skau Colleen T. , Neidt Erin M. , Kovar David R.
Published: February 2009

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.
Boosting for high-dimensional time-to-event data with competing risks
Authors: Binder Harald , Allignol Arthur , Schumacher Martin , Beyersmann Jan
Published: February 2009

Motivation: For analyzing high-dimensional time-to-event data with competing risks, tailored modeling techniques are required that consider the event of interest and the competing events at the same time, while also dealing with censoring. For low-dimensional settings, proportional hazards models for the subdistribution hazard have been proposed, but an adaptation for high-dimensional settings is missing. In addition, tools for judging the prediction performance of fitted models have to be provided.

Results: We propose a boosting approach for fitting proportional subdistribution hazards models for high-dimensional data, that can e.g. incorporate a large number of microarray features, while also taking clinical covariates into account. Prediction performance is evaluated using bootstrap.632+ estimates of prediction error curves, adapted for the competing risks setting. This is illustrated with bladder cancer microarray data, where simultaneous consideration of both, the event of interest and competing events, allows for judging the additional predictive power gained from incorporating microarray measurements.

Availability: The proposed boosting approach is implemented in the R package CoxBoost and prediction error estimation in the package peperr, both available from CRAN.

Contact:  binderh@fdm.uni-freiburg.de
Predicting helix–helix interactions from residue contacts in membrane proteins
Authors: Lo Allan , Chiu Yi-Yuan , Rødland Einar Andreas , Lyu Ping-Chiang , Sung Ting-Yi , Hsu Wen-Lian
Published: February 2009

Motivation: Helix–helix interactions play a critical role in the structure assembly, stability and function of membrane proteins. On the molecular level, the interactions are mediated by one or more residue contacts. Although previous studies focused on helix-packing patterns and sequence motifs, few of them developed methods specifically for contact prediction.

Results: We present a new hierarchical framework for contact prediction, with an application in membrane proteins. The hierarchical scheme consists of two levels: in the first level, contact residues are predicted from the sequence and their pairing relationships are further predicted in the second level. Statistical analyses on contact propensities are combined with other sequence and structural information for training the support vector machine classifiers. Evaluated on 52 protein chains using leave-one-out cross validation (LOOCV) and an independent test set of 14 protein chains, the two-level approach consistently improves the conventional direct approach in prediction accuracy, with 80% reduction of input for prediction. Furthermore, the predicted contacts are then used to infer interactions between pairs of helices. When at least three predicted contacts are required for an inferred interaction, the accuracy, sensitivity and specificity are 56%, 40% and 89%, respectively. Our results demonstrate that a hierarchical framework can be applied to eliminate false positives (FP) while reducing computational complexity in predicting contacts. Together with the estimated contact propensities, this method can be used to gain insights into helix-packing in membrane proteins.

Availability:  http://bio-cluster.iis.sinica.edu.tw/TMhit/

Contact:  tsung@iis.sinica.edu.tw

Supplementary information:  Supplementary data are available at Bioinformatics online.
Shark depredation rates in pelagic longline fisheries: a case study from the Northwest Atlantic
Authors: MacNeil M. Aaron , Carlson John K. , Beerkircher Lawrence R.
Published: February 2009

MacNeil, M. A., Carlson, J. K., and Beerkircher, L. R. 2009. Shark depredation rates in pelagic longline fisheries: a case study from the Northwest Atlantic. – ICES Journal of Marine Science, 66: 708–719. A suite of modelling approaches was employed to analyse shark depredation rates from the US Atlantic pelagic longline fishery. As depredation events are relatively rare, there are a large number of zeroes in pelagic longline data and conventional generalized linear models (GLMs) may be ineffective as tools for statistical inference. GLMs (Poisson and negative binomial), two-part (delta-lognormal and truncated negative binomial, T-NB), and mixture models (zero-inflated Poisson, ZIP, and zero-inflated negative binomial, ZINB) were used to understand the factors that contributed most to the occurrence of depredation events that included a small proportion of whale damage. Of the six distribution forms used, only the ZIP and T-NB models performed adequately in describing depredation data, and the T-NB and ZINB models outperformed the ZIP models in bootstrap cross-validation estimates of prediction error. Candidate T-NB and ZINB model results showed that encounter probabilities were more strongly related to large-scale covariates (space, season) and that depredation counts were correlated with small-scale characteristics of the fishery (temperature, catch composition). Moreover, there was little evidence of historical trends in depredation rates. The results show that the factors contributing to most depredation events are those already controlled by ships' captains and, beyond novel technologies to repel sharks, there may be little more to do to reduce depredation loss in the fishery within current economic and operational constraints.
Thin scattering layers observed by airborne lidar
Authors: Churnside James H. , Donaghay Percy L.
Published: February 2009

Churnside, J. H., and Donaghay, P. L. 2009. Thin scattering layers observed by airborne lidar. – ICES Journal of Marine Science, 66: 778–789. More than 2000 km of thin (<3 m) optical scattering layers were identified in 80 000 km of airborne lidar data collected from a variety of oceanic and coastal waters. The spatial characteristics of thin layers varied dramatically from (i) those that were self-contained features consistently <3–4 m thick over their 1–12 km extent to (ii) those that were clearly parts of much longer layers that had gaps and/or regions where the layer became more intense and much thicker than the 3-m criterion. The characteristics of the lidar signal suggest that plankton was the most likely source of scattering. Examples from upwelling regions, areas with large fresh-water influx, and warm-core eddies are presented. The results are quite consistent with the characteristics observed in studies of thin plankton layers in fjords and near-coastal waters. These layers exhibit great spatial variability that is difficult to observe using traditional methods, and examples of layer perturbations by both linear and non-linear internal waves are presented. The results suggest that airborne lidar can be a powerful tool not only for detecting and mapping the spatial extent of thin scattering layers and linking their occurrence to larger scale physical processes, but also for tracking their evolution over time and guiding the ship-based sampling needed to understand their composition, dynamics, and impacts. Such a capability will be crucial in future studies designed to test the hypothesis that thin plankton layers have the spatial extent and intensity to play a key role in controlling the recruitment of fish larvae, biogeochemical cycling, trophic transfer processes, plankton biodiversity, and harmful algal bloom dynamics.
Antioxidant enzyme activities in maize plants colonized with Piriformospora indica
Authors: Kumar Manoj , Yadav Vikas , Tuteja Narendra , Johri Atul Kumar
Published: February 2009

The bioprotection performance ofPiriformospora indicaagainst the root parasiteFusarium verticillioideswas studied. We found that maize plants first grown withF. verticillioidesand at day 10 inoculated withP. indicashowed improvements in biomass, and root length and number as compared with plants grown withF. verticillioidesalone. To validate our finding that inoculation withP. indicasuppresses colonization byF. verticillioides, we performed PCR analyses usingP. indica- andF. verticillioides-specific primers. Our results showed that inoculation withP. indicasuppresses further colonization byF. verticillioides. We hypothesized that as the colonization byP. indicaincreases, the presence of/colonization byF. verticillioidesdecreases. In roots, catalase (CAT), glutathione reductase (GR), glutathioneS-transferase (GST) and superoxide dismutase (SOD) activities were found to be higher inF. verticillioides-colonized plants than in non-colonized plants. Increased activity of antioxidant enzymes minimizes the chances of oxidative burst (excessive production of reactive oxygen species), and thereforeF. verticillioidesmight be protected from the oxidative defence system during colonization. We also observed decreased antioxidant enzyme activities in plants first inoculated withF. verticillioidesand at day 10 inoculated withP. indicaas compared with plants inoculated withF. verticillioidesalone. These decreased antioxidant enzyme activities due to the presence ofP. indicahelp the plant to overcome the disease load ofF. verticillioides. We propose thatP. indicacan be used as a bioprotection agent against the root parasiteF. verticillioides.
The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization
Authors: Karst Johanna C. , Foucher Anne-Emmanuelle , Campbell Tracey L. , Di Guilmi Anne-Marie , Stroebel David , Mangat Chand S. , Brown Eric D. , Jault Jean-Michel
Published: February 2009

Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study ofBacillus subtilisYdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of theydiBgene yielded a mutant with much reduced growth rate compared to the wild-type strain.In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed aKmof about 60 μM for ATP and aVmaxof about 10 nmol min−1(mg protein)−1(kcat∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain forydiB, showing that the ATPase activity of YdiB is required for thein vivofunction of the protein. Oligomerization was also observed with the purified YjeE fromEscherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in aB. subtilisextract, or when stabilized by formaldehyde cross-linking for YjeE fromE. coli, suggesting that oligomerization might regulate the function of this new class of proteinsin vivo.
Phenotypic characterization and genomic DNA polymorphisms of Escherichia coli strains isolated as the sole micro-organism from vaginal infections
Authors: Lobos Olga , Padilla Carlos
Published: February 2009

Vaginal infections such as vulvovaginal candiadiasis, trichomoniasis and bacterial vaginosis are common worldwide. Accurate diagnosis and prescription of appropriate treatments are important since these infections are linked to adverse outcomes for women during pregnancy and for newborns. Several aetiological agents are responsible for these infectious diseases; however, the presence of Escherichia coli in these infections is controversial. Thus, it is important to identify some phenotypic and genotypic properties of E. coli strains isolated from vaginal infections. Forty-six E. coli strains isolated from vaginal fluid as the sole micro-organism, and 20 other E. coli strains isolated from other samples (urinary tract infections, otitis and septicaemia) were analysed by several phenotypic tests. In addition, genotypic features were studied by RAPD-PCR techniques. Biochemical tests showed that the E. coli strains isolated from vaginal fluid could be grouped into a single cluster which is subdivided into two phenogroups. Analysis of the dendrogram based on fragment length polymorphisms of genomic DNA indicated that E. coli isolates from vaginal infections form a single cluster with two subdivisions. Further studies are needed to analyse the molecular structure and virulence characteristics of these E. coli strains in order to determine their potential role in vaginal infections.
Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath
Authors: Ali Hanif , Murrell J. Colin
Published: February 2009

A series of integrative and versatile broad-host-range promoter-probe vectors carrying reporter genes encoding green fluorescent protein (GFP), catechol 2,3-dioxygenase (XylE) or β-galactosidase (LacZ) were constructed for use in methanotrophs. These vectors facilitated the measurement of in vivo promoter activity in methanotrophs under defined growth conditions. They were tested by constructing transcriptional fusions between the soluble methane monooxygenase (sMMO) σ
54 promoter or particulate methane monooxygenase (pMMO) σ
70 promoter from Methylococcus capsulatus and the reporter genes. Reporter gene activity was measured under high- and low-copper growth conditions and the data obtained closely reflected transcriptional regulation of the sMMO or pMMO operon, thus demonstrating the suitability of these vectors for assessing promoter activity in methanotrophs. When β-galactosidase expression was coupled with the fluorogenic substrate 4-methylumbelliferyl β-d-glucuronide it yielded a sensitive and powerful screening system for detecting cells expressing this reporter gene. These data were substantiated with independent experiments using RT-PCR and RNA dot-blot analysis.
The wzm gene located on the pRhico plasmid of Azospirillum brasilense Sp7 is involved in lipopolysaccharide synthesis
Authors: Lerner Anat , Okon Yaacov , Burdman Saul
Published: February 2009

Several genes involved in the interaction between Azospirillum brasilense Sp7 and plants are located on the pRhico plasmid. Here we report the characterization of an Sp7 mutant strain with impairment of the pRhico-located gene wzm. This gene encodes an inner-membrane component of an ATP-binding cassette (ABC) transporter with similarity to transporters involved in surface polysaccharide export. Indeed, SDS-PAGE revealed that LPS synthesis is affected in the wzm mutant. No significant differences were observed between wild-type and mutant strains in exopolysaccharide (EPS) amount; however, several differences were observed between them in EPS monosaccharide composition, and only wild-type colonies stained positively with Congo red. Microscopy revealed that wzm mutant cells are longer and thinner, and exhibit several differences in their cell surface relative to the wild-type. The wzm mutant was more resistant to oxidative stress, starvation, desiccation, heat and osmotic shock than the wild-type. In contrast, the mutant was more susceptible than the wild-type to UV radiation and saline stress. The strains also differed in their susceptibility to different antibiotics. Differences between the strains were also observed in their outer-membrane protein composition. No differences were observed between strains in their ability to attach to sweet corn roots and seeds, and to promote growth under the tested conditions. As LPS plays an important role in cell envelope structural integrity, we propose that the pleiotropic phenotypic changes observed in the wzm mutant are due to its altered LPS relative to the wild-type.
lmo1273, a novel gene involved in Listeria monocytogenes virulence
Authors: Bigot Armelle , Raynaud Catherine , Dubail Iharilalao , Dupuis Marion , Hossain Hamid , Hain Torsten , Chakraborty Trinad , Charbit Alain
Published: February 2009

Listeria monocytogenesis a foodborne pathogen able to infect humans and many other mammalian species, leading to serious, often fatal disease. We have previously identified a five-gene locus in the genome ofL. monocytogenesEGD-e which comprised three contiguous genes encoding paralogous type I signal peptidases. In the present study, we focused on the two distal genes of the locus (lmo1272andlmo1273), encoding proteins sharing significant similarities with the YlqF and RnhB proteins, respectively, ofBacillus subtilis.lmo1273could complement anEscherichia coli rnhA-rnhBthermosensitive growth phenotype, suggesting that it encodes a functional RNase H. Strikingly, inactivation oflmo1273provoked a strong attenuation of virulence in the mouse model, and kinetic studies in infected mice revealed that multiplication of thelmo1273mutant in target organs was significantly impaired. However, the mutation did not impairL. monocytogenesintracellular multiplication or cell-to-cell spread in cell culture models. Transcriptional profiles obtained with anlmo1273-overexpressing strain were compared to those of the wild-type strain, using microarray analyses. The data obtained suggest a pleiotropic regulatory role of Lmo1273 and possible links with amino acid uptake.
Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors
Authors: Dekimpe Valérie , Déziel Eric
Published: February 2009

Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C4-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C12-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.
Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection
Authors: Lewis Claire , Skovierova Henrieta , Rowley Gary , Rezuchova Bronislava , Homerova Dagmar , Stevenson Andrew , Spencer Janice , Farn Jacinta , Kormanec Jan , Roberts Mark
Published: February 2009

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ
E (RpoE). S. Typhimurium htrA also possesses a σ
E-dependent promoter; however, we found that the absence of σ
E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ
E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ
70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.
Short motif sequences determine the targets of the prokaryotic CRISPR defence system
Authors: Mojica F. J. M. , Díez-Villaseñor C. , García-Martínez J. , Almendros C.
Published: February 2009

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) ofStreptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.
Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences
Authors: Watanabe Keiro , Tsuchida Yoshiki , Okibe Naoko , Teramoto Haruhiko , Suzuki Nobuaki , Inui Masayuki , Yukawa Hideaki
Published: February 2009

Systematic screening of secretion proteins using an approach based on the completely sequenced genome ofCorynebacterium glutamicumR revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-formα-amylase derived fromGeobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secretedα-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency.
Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis
Authors: Yoshida Yasuo , Sasaki Takako , Ito Shuntaro , Tamura Haruki , Kunimatsu Kazushi , Kato Hirohisa
Published: February 2009

Indole produced via theβ-elimination reaction ofl-tryptophan by pyridoxal 5′-phosphate-dependent tryptophanase (EC 4.1.99.1) has recently been shown to be an extracellular and intercellular signalling molecule in bacteria, and controls bacterial biofilm formation and virulence factors. In the present study, we determined the molecular basis of indole production in the periodontopathogenic bacteriumPorphyromonas gingivalis. A database search showed that the amino acid sequence deduced frompg1401ofP. gingivalisW83 is 45 % identical with that fromtnaAofEscherichia coliK-12, which encodes tryptophanase. Replacement of thepg1401gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction ofpg1401, demonstrating that the gene is functionally equivalent totnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlikeE. coli tnaA,pg1401is expressed alone inP. gingivalisand that the nucleotide sequence of the transcription start site is different, suggesting that the expression ofP. gingivalis tnaAis controlled by a unique mechanism. Purified recombinantP. gingivalistryptophanase exhibited the Michaelis–Menten kinetics valuesKm=0.20±0.01 mM andkcat=1.37±0.06 s−1in potassium phosphate buffer, but in sodium phosphate buffer, the enzyme showed lower activity. However, the cation in the buffer, K+or Na+, did not appear to affect the quaternary structure of the enzyme or the binding of pyridoxal 5′-phosphate to the enzyme. The enzyme also degradedS-ethyl-l-cysteine andS-methyl-l-cysteine, but notl-alanine,l-serine orl-cysteine.
Evaluation of procedures for outer membrane isolation from Campylobacter jejuni
Authors: Hobb Rhonda I. , Fields Joshua A. , Burns Christopher M. , Thompson Stuart A.
Published: February 2009

Although infection withCampylobacter jejuniis one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a ‘gold standard’ method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outer-membrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30–60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g.Escherichia coliorSalmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes usingN-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifyingC. jejunisurface proteins as vaccine components.
The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa
Authors: Gooderham W. James , Gellatly Shaan L. , Sanschagrin François , McPhee Joseph B. , Bains Manjeet , Cosseau Celine , Levesque Roger C. , Hancock Robert E. W.
Published: February 2009

Pseudomonas aeruginosais a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation ofphoQcaused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.
Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines
Authors: Zhou Xiaohui , Konkel Michael E. , Call Douglas R.
Published: February 2009

TheVibrio parahaemolyticustype III secretion system 1 (T3SS1) induces cytotoxicity in mammalian epithelial cells. We characterized the cell death phenotype in both epithelial (HeLa) and monocytic (U937) cell lines following infection withV. parahaemolyticus. Using a combination of the wild-type strain and gene knockouts, we confirmed thatV. parahaemolyticusstrain NY-4 was able to induce cell death in both cell lines via a T3SS1-dependent mechanism. Bacterial contact, but not internalization, was required for T3SS1-induced cytotoxicity. The mechanism of cell death involves formation of a pore structure on the surface of infected HeLa and U937 cells, as demonstrated by cellular swelling, uptake of cell membrane-impermeable dye and protection of cytotoxicity by osmoprotectant (PEG3350). Western blot analysis showed that poly ADP ribose polymerase (PARP) was not cleaved and remained in its full-length active form. This result was evident for seven differentV. parahaemolyticusstrains.V. parahaemolyticus-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) or the caspase-1 inhibitorN-acetyl-tyrosyl-valyl-alanyl-aspartyl-aldehyde (Ac-YVAD-CHO); thus, caspases were not involved in T3SS1-induced cytotoxicity. DNA fragmentation was not evident following infection and autophagic vacuoles were not observed after monodansylcadaverine staining. We conclude that T3SS1 ofV. parahaemolyticusstrain NY-4 induces a host cell death primarily via oncosis rather than apoptosis, pyroptosis or autophagy.