Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. campestris

Background  

Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli.     

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.     

Methacarn (methanol-Carnoy) fixation

Summary According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO₄ cause considerable loss of helix (22–29% and 39–66% respectively). KMnO₄ and glutaraldehyde followed by OsO₄ produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.     

Strategic egg allocation in the zebra fish, Danio rerio

Females across a range of taxa have been shown to differentiallyallocate their reproductive resources according to the attractivenessof their mate. Previous studies demonstrated a female preferencefor larger males in the zebra fish but have so far failed touncover a size-mediated difference in male mating success, possiblydue to the effects of male–male competition. By controllingfor male–male competition in the present study, we showthat females strategically allocate their reproductive resources(i.e., eggs) toward larger males. When females were mated sequentiallywith a large and small male, they released a greater numberof eggs to the second male when he was large than when he wassmall. Furthermore, there was also a trend for females to releasea greater proportion of their eggs to the first male when hewas large. Across females, the total number of eggs laid byeach female increased with the average standard length of themale pair, whereas the number of eggs laid to the second malealso increased with his standard length. This study representsone of the first attempts at identifying differential allocationin a resource-free egg scatterer and suggests that female preferencesmay play a greater role in the reproductive success of malesin this species than previously envisaged.     

Intravenous transfusion of BCR-activated B cells protects NOD mice from type 1 diabetes in an IL-10-dependent manner

Although B cells play a pathogenic role in the initiation of type 1 diabetes (T1D) in NOD mice, it is not known whether activated B cells can maintain tolerance and transfer protection from T1D. In this study, we demonstrate that i.v. transfusion of BCR-stimulated NOD spleen B cells into NOD mice starting at 5-6 wk of age both delays onset and reduces the incidence of T1D, whereas treatment initiated at 9 wk of age only delays onset of T1D. This BCR-activated B cell-induced protection from T1D requires IL-10 production by B cells, as transfusion of activated B cells from NOD.IL-10(-/-) mice does not confer protection from T1D. Consistent with this result, severe insulitis was observed in the islets of NOD recipients of transfused NOD.IL-10(-/-) BCR-stimulated B cells but not in the islets of NOD recipients of transfused BCR-stimulated NOD B cells. The therapeutic effect of transfused activated NOD B cells correlates closely with the observed decreased islet inflammation, reduced IFN-gamma production and increased production of IL-4 and IL-10 by splenocytes and CD4(+) T cells from NOD recipients of BCR-stimulated NOD B cells relative to splenocytes and CD4(+) T cells from PBS-treated control NOD mice. Our data demonstrate that transfused BCR-stimulated B cells can maintain long-term tolerance and protect NOD mice from T1D by an IL-10-dependent mechanism, and raise the possibility that i.v. transfusion of autologous IL-10-producing BCR-activated B cells may be used therapeutically to protect human subjects at risk for T1D.     

A method to generate recombinant <Emphasis Type="Italic">Salmonella typhi</Emphasis> Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated ‘targeting cassettes’ that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking ‘arms’ of sequence (ca. 1,000 bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains.     

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.     

The Drosophila PNG kinase complex regulates the translation of cyclin B

The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.     

Quantifying human disturbance on antipredator behavior and flush initiation distance in yellow-bellied marmots

Human disturbance may differentially affect the behavior of wild animals and such behavioral perturbations may have fitness consequences. To understand the effects of specific types of human disturbance on antipredator behavior, a behavior whose performance enhances survival, we studied yellow-bellied marmots (Marmota flaviventris). We quantified both antipredator vigilance and the flight initiation distance of the marmots to an approaching human in six different colony sites where we also quantified the frequency and type of human visitation. We developed an analysis framework, using linear mixed models, and found that: (1) when the presence of motorized vehicles and bicycles was high, marmots increased the proportion of time spent vigilant (pseudo R² = 0.33 and 0.31 for motorized vehicles and bicycles, P < 0.05) and decreased the time spent foraging (pseudo R² = 0.29 and 0.23 for motorized vehicles and bicycles, P < 0.05), (2) there was no significant effect of the presence of pedestrians on the time allocated to vigilance and foraging (pseudo R² = 0.25 and 0.19, P > 0.05), (3) marmots decreased the flight initiation distance as disturbance of motorized vehicles (pseudo R² = 0.85) and pedestrians (pseudo R² = 0.84) increased (P < 0.05), and (4) when we considered bicycles as the disturbance, juveniles tolerated closer approaches than adults or yearlings (P < 0.001). Marmots thus responded to some human disturbance by adjusting time spent in foraging and shortening the tolerance distance. Since these behavioral responses could have significant implications for survival and reproduction, we should generally view human disturbance as something that can influence natural antipredator behavior. Importantly, based on an understanding of the differential effects of human activities on wildlife, reducing human disturbance should be taken into account for wildlife management. In addition, our approach will be useful to quantify differential effects of humans on wildlife and to enhance our ability to manage those impacts.