Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.     

NK cell responses to Plasmodium infection and control of intrahepatic parasite development

Various components of innate and adaptive immunity contribute to host defenses against Plasmodium infection. We investigated the contribution of NK cells to the immune response to primary infection with Plasmodium yoelii sporozoites in C57BL/6 mice. We found that hepatic and splenic NK cells were activated during infection and displayed different phenotypic and functional properties. The number of hepatic NK cells increased whereas the number of splenic NK cells decreased. Expression of the Ly49 repertoire was modified in the spleen but not in the liver. Splenic and hepatic NK cells have a different inflammatory cytokines profile production. In addition, liver NK cells were cytotoxic to YAC-1 cells and P. yoelii liver stages in vitro but not to erythrocytic stages. No such activity was observed with splenic NK cells from infected mice. These in vitro results were confirmed by the in vivo observation that Rag2(-/-) mice were more resistant to sporozoite infection than Rag2(-/-) gamma c(-/-) mice, whereas survival rates were similar for the two strains following blood-stage infection. Thus, NK cells are involved in early immune mechanisms controlling Plasmodium infection, mostly at the pre-erythrocytic stage.     

Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov.

Three Gram-positive bacterial strains, 7-3, 255-15 and 190-11, previously isolated from Siberian permafrost, were characterized and taxonomically classified. These microorganisms are rod-shaped, facultative aerobic, motile with peritrichous flagella and their growth ranges are from -2.5 to 40 degrees C. The chemotaxonomic markers indicated that the three strains belong to the genus Exiguobacterium. Their peptidoglycan type was A3alpha L-Lys-Gly. The predominant menaquinone detected in all three strains was MK7. The polar lipids present were phosphatidyl-glycerol, diphosphatidyl-glycerol and phosphatidyl-ethanolamine. The major fatty acids were iso-C13:0, anteiso-C13:0, iso-C15:0, C16:0 and iso-C17:0. Phylogenetic analysis based on 16S rRNA and six diverse genes, gyrB (gyrase subunit B), rpoB (DNA-directed RNA polymerase beta subunit), recA (homologous recombination), csp (cold shock protein), hsp70 (ClassI-heat shock protein-chaperonin) and citC (isocitrate dehydrogenase), indicated that the strains were closely related to Exiguobacterium undae (DSM 14481(T)) and Exiguobacterium antarcticum (DSM 14480(T)). On the basis of the phenotypic characteristics, phylogenetic data and DNA-DNA reassociation data, strain 190-11 was classified as E. undae, while the other two isolates, 7-3 and 255-15, comprise a novel species, for which the name Exiguobacterium sibiricum sp. nov. is proposed.     

Use of competitive DNA hybridization to identify differences in the genomes of bacteria

Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we report the development and testing of a biochemical approach for targeted sequencing of only those chromosomal regions that differ between two DNA preparations. The method, designated GFE (genome fragment enrichment) uses competitive solution hybridization and positive selection to obtain genomic DNA fragments that are present in one pool of fragments but not another. Repeated comparisons of the genomes of Enterococcus faecalis and E. faecium led to the identification of 225 putative genome-specific DNA fragments. Species and strain variations within these fragments were confirmed by both experimental and bioinformatic analyses. The E. faecalis genome-specific sequences identified included both a preponderance of those predicted to encode surface-exposed proteins, as well as several previously described unique marker regions embedded within highly conserved rrn operons. The GFE strategy we describe efficiently identified genomic differences between two enterococcal genomes, and will be widely applicable for studying genetic variation among closely related bacterial species.     

Influence of Hydrological Pulse on Bacterial Growth and DOC Uptake in a Clear-Water Amazonian Lake

This study was conducted to evaluate: (1) the bacterial growth and the dissolved organic carbon (DOC) uptake in an Amazonian lake (Lake Batata) at high-water and low-water periods of the flood pulse; (2) the influence of nitrogen and phosphorus (NP) additions on bacterial growth and DOC uptake in Lake Batata at two flood pulse periods; and (3) the bioavailability of the main DOC sources in Lake Batata. Lake Batata is a typical clear-water Amazonian lake, located in the watershed of Trombetas River, Central Amazon, Brazil. Bacterial batch cultures were set up with 90% 0.2-μm filtered water and 10% inoculum from Lake Batata. N-NH₄NO₃ and P-KH₂PO₄, with final concentrations of 50 and 5 μM, respectively, were added to the cultures, except for controls. Extra sources of DOC (e.g., algal lysate, plant leachates) were added to constitute six distinct treatments. Bacterial response was measured by maximum bacterial abundance and rates of bacterial production, respiration, DOC uptake, and bacterial growth efficiency (BGE). Bacterial growth and DOC uptake were higher in NP treatments than in controls, indicating a consistent nutrient limitation in Lake Batata. The composition of DOC also seems to be an important regulating factor of bacterial growth in Lake Batata. Seasonally, bacterial growth and DOC bioavailability were higher at low-water period, when the phytoplankton is a significant extra source of DOC, than at high-water period, when the forest is the main source of DOC. DOC bioavailability was better estimated based on the diversity and the diagenetic stage of carbon compounds than on single classes of labile compounds. Changes in BGE were better related to CNP stoichiometry in the water, and the “excess” of organic substrates was oxidized in catabolism, despite the quality of these compounds for bacterial growth. Finally, we conclude that bacterial growth and DOC uptake vary throughout the flood pulse in clear-water Amazonian ecosystems as a result of changes in nutrient concentration and in DOC composition.     

Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.     

Remodeling of the epitope repertoire of a candidate idiotype vaccine by targeting to lysosomal degradation in dendritic cells

The generation of efficacious vaccines against self-antigens expressed in tumor cells requires breakage of tolerance, and the refocusing of immune responses toward epitopes for which tolerance may not be established. While the presentation of tumor antigens by mature dendritic cells (mDC) may surpass tolerance, broadening of the antigenic repertoire remains an issue. We report that fusion of the candidate idiotype vaccine IGKV3-20 to the Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen (EBNA)-1 inhibits degradation by the proteasome and redirects processing to the lysosome. mDCs transduced with a recombinant lentivirus encoding the chimeric idiotype efficiently primed CD4+ and CD8+ cytotoxic T-cell (CTL) responses that lysed autologous blasts expressing IGKV3-20 or pulsed with IGKV3-20 synthetic peptides, and HLA-matched IGKV3-20-positive tumor cell lines. Comparison of the cytotoxic response of CD4+ and CD8+ T lymphocytes activated by mDCs expressing the wild-type or chimeric IGKV3-20 reveled largely non-overlapping epitope repertoires in both CD4+ and CD8+ effectors. Thus, fusion to the GAr may provide an effective means to broaden the immune response to an endogenous protein by promoting the presentation of antigenic epitopes that require a lysosome-dependent processing step.     

Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids

Over the next decade, wheat grain production must increase to meet the demand of a fast growing human population. One strategy to meet this challenge is to raise wheat productivity by optimizing plant stature. The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the Green Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve lodging resistance, without compromising grain yield. Rht8 is widely used in dry environments such as Mediterranean countries where it increases plant adaptability. With recent climate change, its use could become increasingly important even in more northern latitudes. In the present study, the characterization of Rht8 was furthered. Morphological analyses show that the semi-dwarf phenotype of Rht8 lines is due to shorter internodal segments along the wheat culm, achieved through reduced cell elongation. Physiological experiments show that the reduced cell elongation is not due to defective gibberellin biosynthesis or signalling, but possibly to a reduced sensitivity to brassinosteroids. Using a fine-resolution mapping approach and screening 3104 F(2) individuals of a newly developed mapping population, the Rht8 genetic interval was reduced from 20.5 cM to 1.29 cM. Comparative genomics with model genomes confined the Rht8 syntenic intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of Brachypodium distachyon chromosome 5. The very high resolution potential of the plant material generated is crucial for the eventual cloning of Rht8.