Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination

The processes leading to systemic dissemination of the obligate intracellular parasite Toxoplasma gondii remain unelucidated. In vitro studies on human and murine dendritic cells (DC) revealed that active invasion of DC by Toxoplasma induces a state of hypermotility in DC, enabling transmigration of infected DC across endothelial cell monolayers in the absence of chemotactic stimuli. Infected DC exhibited upregulation of maturation markers and co-stimulatory molecules. While modulation of cell adhesion molecules CD11/CD18 was similar for Toxoplasma-infected DC and lipopolysaccharide (LPS)-matured DC, Toxoplasma-infected DC did not exhibit upregulation of CD54/ICAM-1. Induction of host cell migration in vitro required live intracellular parasite(s) and was inhibited by uncoupling the Gi-protein signalling pathway with pertussis toxin, but did not depend on CCR5, CCR7 or Toll/interleukin-1 receptor signalling. When migration of Toxoplasma-infected DC was compared with migration of LPS-stimulated DC in vivo, similar or higher numbers of Toxoplasma-infected DC reached the mesenteric lymph nodes and spleen respectively. Adoptive transfer of Toxoplasma-infected DC resulted in more rapid dissemination of parasites to distant organs and in exacerbation of infection compared with inoculation with free parasites. Altogether, these findings show that Toxoplasma is able to subvert the regulation of host cell motility and likely exploits the host's natural pathways of cellular migration for parasite dissemination.     

Characterization of the endopeptidase activity of tripeptidyl-peptidase II

Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.     

Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction

Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function.     

Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC-ESI-Q-TOF MS/MS. Ninety of a total of 248 identified unique proteins were predicted to possess transmembrane segments (TMSs), and 56 of these have more than one TMS. Seventy-nine of the identified proteins are annotated to be involved in transport of amino acids, oligosaccharides, inorganic ions, nucleotides, phosphate or exopolysaccharides, or to belong to the F1F0-ATP-synthetase complex and the protein translocation machinery, respectively.     

Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr

There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D(2)O) and sucrose. An advantage of the D(2)O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D(2)O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.     

Proteomic analysis of the cyanobacterium of the Azolla symbiosis: identity, adaptation, and NifH modification

Cyanobacteria are able to form stable nitrogen-fixing symbioseswith diverse eukaryotes. To extend our understanding of adaptationsimposed by plant hosts, two-dimensional gel electrophoresisand mass spectrometry (MS) were used for comparative proteinexpression profiling of a cyanobacterium (cyanobiont) dwellingin leaf cavities of the water-fern Azolla filiculoides. Homology-basedprotein identification using peptide mass fingerprinting [matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF-MS)],tandem MS analyses, and sequence homology searches resultedin an identification success rate of 79% of proteins analysedin the unsequenced cyanobiont. Compared with a free-living strain,processes related to energy production, nitrogen and carbonmetabolism, and stress-related functions were up-regulated inthe cyanobiont while photosynthesis and metabolic turnover rateswere down-regulated, stressing a slow heterotrophic mode ofgrowth, as well as high heterocyst frequencies and nitrogen-fixingcapacities. The first molecular data set on the nature of theNifH post-translational modification in cyanobacteria was alsoobtained: peptide mass spectra of the protein demonstrated thepresence of a 300–400 Da protein modification localizedto a specific 13 amino acid sequence, within the part of theprotein that is ADP-ribosylated in other bacteria and closeto the active site of nitrogenase. Furthermore, the distributionof the highest scoring database hits for the identified proteinspoints to the possibility of using proteomic data in taxonomy. Key words: Azolla, cyanobacteria, NifH modification, proteomics, symbiosis, taxonomy Received 24 June 2007; Revised 21 October 2007 Accepted 22 October 2007     

Effects of calcium ion concentration on starch hydrolysis of barley alpha-amylase isozymes

Barley alpha-amylase genes, amy1 and amy2, were separately cloned into the expression vector of pPICZalphaA and recombinant Pichia strains were established by homologous recombination. Both AMYs from Pichia shared almost identical hydrolysis patterns on short maltooligosaccharides to result in glucose, maltose, or maltotriose. Against insoluble blue starch, AMY1 showed the highest activity at 0.1-5 mM calcium concentration, whereas 15-20 mM was optimal for AMY2. On the hydrolysis of soluble starch, unexpectedly, there was no significant difference between AMYs with increase of calcium. However, the relative activity on various starch substrates was significantly different between AMYs, which supports that the isozymes are clearly distinguished from each other on the basis of their unique preferences for substrates.     

Temporal fluctuations of Y-chromosomal variation in Bos taurus

Phylogeography has recently become more abundant in studies of demographic history of both wild and domestic species. A single nucleotide polymorphism (SNP) in the intron of the Y-chromosomal gene UTY19 displays a north-south gradient in modern cattle. Support for this geographical distribution of haplogroups has previously also been seen in ancient cattle from Germany. However, when analysing 38 historic remains of domestic bulls and three aurochs from northern Europe for this SNP we found no such association. Instead, we noted extensive amounts of temporal variation that can be attributed to transportation of cattle and late breed formation.     

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements

Arginine257 (R257), in the de-helix that caps the Q(B) site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the Q(B) site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced Q(B), Q(B)(-) ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q(A)(-) to Q(B)) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 microM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q(B)(-) with S(2) was reduced by 20-40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of Q(B)/Q(B)(-). Further, since the recombination of Q(A)(-) with S(2) was unaffected, we suggest that no significant change in redox potential of Q(A)/Q(A)(-) occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the Q(B) site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the Q(B)/Q(B)(-) pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.     

Simulated climate change impacts on fluxes of carbon in Norway spruce ecosystems along a climatic transect in Sweden

A simulation study based on recent regional climate scenarios for Sweden investigated possible changes in carbon (C) dynamics and net ecosystem exchange (NEE) of Swedish Norway spruce forest ecosystems. Four sites, representative of well-drained soils in four regions, were included. Stand development was simulated for a 100-year rotation period using a coupled model describing abiotic and biotic processes in the soil-plant-atmosphere system. Two IPCC climate change scenarios, corresponding to a mean annual temperature increase of about 2°C (A2) or 3°C (B2) from the reference period 1961–1990 to a new period 2061–2090, were considered. Annual maximum snow depth decreased with the increase in air temperature, whereas maximum soil frost depth and mean annual soil temperature showed only small changes, especially for the sites in northern Sweden. Simulations suggested that in the warmer climate, gross primary production (GPP) increased by 24–32% in northern Sweden and by 32–43% in the south. In the north, the increase was related to the combined effect of air and soil temperature extending the growing season, whereas in the south it was mainly governed by increased N availability due to increased soil temperature. NEE increased by about 20% (A2) or 25% (B2) at all sites, more or less solely due to increased accumulation of C in the tree biomass (including harvest residues), since changes in soil C were small compared with the current climate. Both light use efficiency and water use efficiency were improved in the future climate scenarios, despite increases in atmospheric CO₂ not being considered.