Natural killer cells and innate immunity to protozoan pathogens

Natural killer (NK) cells are lymphoid cells that mediate significant cytotoxic activity and produce high levels of pro-inflammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte-macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-gamma (IFN-gamma) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity.     

Transposase A binding sites in the attachment sites of bacteriophage Mu that are essential for the activity of the enhancer and A binding sites that promote transposition towards Fpro-lac.

In this paper we determine which of the A binding sites in the attachment sites of phage Mu are required for the stimulatory activity of the transpositional enhancer (IAS). For this purpose the transposition frequencies of mini-Mu's with different truncated attachment sites to an Ftet target were measured both in the presence and the absence of the IAS. The results show that in our in vivo assay the L3 and R3 sites are dispensable for functioning of the IAS. An additional deletion of L2 or R2 however abolishes the stimulating activity of the enhancer suggesting an interaction between A molecules bound to these sites and the IAS. The residual transposition activity of a IAS-containing mini Mu in which R2 (and R3) are deleted is much lower than the activity of the comparable construct without the IAS. This means that in the absence of R2 the IAS is inhibiting transposition. Such an inhibition is not observed when L2 (and L3) are deleted. This suggests that the IAS interacts with the attachment sites in an ordered fashion, first with attL and then with attR. Furthermore we show that mini-Mu transposition is enhanced when Fpro-lac is used as a target instead of Ftet. We show that this elevated transposition is dependent on the Mu A binding sites L2,L3 and R2. These sequences could possibly mediate an interaction between the mini-Mu plasmid and sequences present on Fpro-lac.     

A critical evaluation of the Oscillayers methods and datasets

Here we evaluate Oscillayers, a new method that aims to estimate palaeoclimates for the past 5.4 Myr, and discuss the associated theoretical and methodological issues. We show that the theoretical foundation of Oscillayers is inherently limited, because the method cannot incorporate spatio‐temporal variation and different forcing mechanisms into climate reconstructions. In addition, several methodological weaknesses are clarified that entrench the palaeoclimatic reconstruction of Oscillayers to patterns of climate change observed between the Last Glacial Maximum and current climates. We test the utility of the Oscillayers method to produce palaeoclimatic reconstructions that are similar to general circulation model (GCM)‐based estimates. On average, only 55.6% of values in the mean annual temperature datasets across the Pliocene and Pleistocene were within ±3°C when compared with corresponding GCM‐based datasets. Furthermore, on average only 75.3% of values in the mean annual precipitation datasets across the Pliocene and Pleistocene were within ±200 mm of rainfall of the GCM‐based estimates. Our results demonstrate that the Oscillayers approach does not provide a robust approximation of palaeoclimatic conditions throughout the Plio‐Pleistocene. Thus, when these datasets are used for scientific analyses, the results should be interpreted with a full appreciation of their limitations, particularly for periods outside the last glacial cycle.     

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

Arsenite oxidase,an ancient bioenergetic enzyme

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

Effects of aluminum sorption on calcium-polygalacturonate network used as soil-root interface model

The objective of this study is to determine the influence of aluminum sorption on a calcium-polygalacturonate (Ca-PG) network used as a soil-root interface model. The Ca-PG network is exposed to aluminum solutions at different concentrations (25-800 microM) at pH 3.50. High concentrations lead to a release of calcium (80%) and aluminum becomes the predominant reticulating cation of the polygalacturonic chains. The FTIR spectra show how aluminum sorption induces shifts of the characteristic bands of carbohydrates in the spectral regions of 1700-1400 and 1200-800 cm(-1), which are enhanced by decreasing intensities. This might be induced by a weakening of the metal-PG complex through conformational variations of the structure. Scanning electron micrographs also show a collapse of the fibrillar structure of Ca-PG that is due to aluminum sorption. This structural rearrangement suggests that the soil-root interface could modify its functionality, affecting the transport of metal ions (nutrients) across the interface and consequently through the cell membranes.