Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Density gradient analysis of DNA replicated during Bacillus subtilis sporulation.

Density gradient centrifugation was used to monitor DNA replication during sporulation of a 5-bromo-2'-deoxyuridine-tolerant, thymidine-requiring strain of Bacillus subtilis. DNA of heavy, intermediate, and light density was found in cells induced to sporulate in the presence of bromodeoxyuridine, but only intermediate DNA was detected in mature spores. Cells grown with bromodeoxyuridine until DNA was in the heavy form formed spores containing intermediate and light DNA when sporulated with thymidine alone.     

The proteins encoded by the rbs operon of Escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC.

Chimeric genes encoding full-length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly-His leader peptide was incorporated so that Ni-chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa-cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly-His leader was also constructed and expressed. Both the chimeric RbsA-C species and the poly-HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.     

A Highlights from MBoC Selection: Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.     

Nod2 Activates NF-kB in CD4+ T Cells but Its Expression Is Dispensable for T Cell-Induced Colitis

Although the etiology of Crohn''s disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4⁺ T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4⁺ T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4⁺ T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3⁺ Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3⁺ Treg cells after OVA feeding. In vitro, CD25⁺ Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4⁺ T cells but its expression is dispensable for the T cell regulation of colitis.     

An IDEA for Short Term Outbreak Projection: Nearcasting Using the Basic Reproduction Number

Background

Communicable disease outbreaks of novel or existing pathogens threaten human health around the globe. It would be desirable to rapidly characterize such outbreaks and develop accurate projections of their duration and cumulative size even when limited preliminary data are available. Here we develop a mathematical model to aid public health authorities in tracking the expansion and contraction of outbreaks with explicit representation of factors (other than population immunity) that may slow epidemic growth.

Methodology

The Incidence Decay and Exponential Adjustment (IDEA) model is a parsimonious function that uses the basic reproduction number R₀, along with a discounting factor to project the growth of outbreaks using only basic epidemiological information (e.g., daily incidence counts).

Principal Findings

Compared to simulated data, IDEA provides highly accurate estimates of total size and duration for a given outbreak when R₀ is low or moderate, and also identifies turning points or new waves. When tested with an outbreak of pandemic influenza A (H1N1), the model generates estimated incidence at the i+1^th serial interval using data from the i^th serial interval within an average of 20% of actual incidence.

Conclusions and Significance

This model for communicable disease outbreaks provides rapid assessments of outbreak growth and public health interventions. Further evaluation in the context of real-world outbreaks will establish the utility of IDEA as a tool for front-line epidemiologists.     

Risk Perception and Risk-Taking Behaviour during Adolescence: The Influence of Personality and Gender

This study investigated the influence of personality characteristics and gender on adolescents’ perception of risk and their risk-taking behaviour. Male and female participants (157 females: 116 males, aged 13–20) completed self-report measures on risk perception, risk-taking and personality. Male participants perceived behaviours as less risky, reportedly took more risks, were less sensitive to negative outcomes and less socially anxious than female participants. Path analysis identified a model in which age, behavioural inhibition and impulsiveness directly influenced risk perception, while age, social anxiety, impulsiveness, sensitivity to reward, behavioural inhibition and risk perception itself were directly or indirectly associated with risk-taking behaviour. Age and behavioural inhibition had direct relationships with social anxiety, and reward sensitivity was associated with impulsiveness. The model was representative for the whole sample and male and female groups separately. The observed relationship between age and social anxiety and the influence this may have on risk-taking behaviour could be key for reducing adolescent risk-taking behaviour. Even though adolescents may understand the riskiness of their behaviour and estimate their vulnerability to risk at a similar level to adults, factors such as anxiety regarding social situations, sensitivity to reward and impulsiveness may exert their influence and make these individuals prone to taking risks. If these associations are proven causal, these factors are, and will continue to be, important targets in prevention and intervention efforts.