Effects of six years atmospheric CO2 enrichment on plant,soil, and soil microbial C of a calcareous grassland

Stimulated plant production and often even larger stimulation of photosynthesis at elevated CO₂ raise the possibility of increased C storage in plants and soils. We analysed ecosystem C partitioning and soil C fluxes in calcareous grassland exposed to elevated CO₂ for 6 years. At elevated CO₂, C pools increased in plants (+23%) and surface litter (+24%), but were not altered in microbes and soil organic matter. Soils were fractionated into particle size and density separates. The amount of low-density macroorganic C, an indicator of particulate soil C inputs from root litter, was not affected by elevated CO₂. Incorporation of C fixed during the experiment (C_new) was tracked by C isotopic analysis of soil fractions which were labelled due to ¹³C depletion of the commercial CO₂ used for atmospheric enrichment. This data constrains estimates of C sequestration (absolute upper bound) and indicates where in soils potentially sequestered C is stored. C_new entered soils at an initial rate of 210±42 g C m^–2 year^–1, but only 554±39 g C_new m^–2 were recovered after 6 years due to the low mean residence time of 1.8 years. Previous process-oriented measurements did not indicate increased plant–soil C fluxes at elevated CO₂ in the same system (¹³C kinetics in soil microbes and fine roots after pulse labelling, and minirhizotron observations). Overall experimental evidence suggests that C storage under elevated CO₂ occurred only in rapidly turned-over fractions such as plants and detritus, and that potential extra soil C inputs were rapidly re-mineralised. We argue that this inference does not conflict with the observed increases in photosynthetic fixation at elevated CO₂, because these are not good predictors of plant growth and soil C fluxes for allometric reasons. C sequestration in this natural system may also be lower than suggested by plant biomass responses to elevated CO₂ because C storage may be limited by stabilisation of C_new in slowly turned-over soil fractions (a prerequisite for long-term storage) rather than by the magnitude of C inputs per se.     

IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPS(d), although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-kappa B, which is shown by an NF-kappa B-driven reporter gene construct. In the presence of an inhibitor of NF-kappa B activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-kappa B drives the expression of IL-9 via three NF-kappa B binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.     

Implicit learning and implicit memory for odors: the influence of odor identification and retention time

One hundred and fifty-two subjects, divided into eight groups, were exposed to a room with a low concentration of either orange or lavender and to an odorless room. In a careful double-blind procedure, neither the subjects nor the experimenters were made aware of the presence of the odors in the experimental conditions. Later they were asked to indicate how well each of 12 odor stimuli, including the experimental and control odors, befitted each of 12 visual contexts, including the exposure rooms. At the end of this session they rated the pleasantness and the familiarity of the odors, and identified them by name. Finally they were debriefed and asked specifically whether they had perceived the experimental odors anywhere in the building. The results of four subjects who answered positively to the latter question were omitted. The results confirm the earlier finding that non-identifiers implicitly link odor and exposure room, whereas identifiers do not show such a link. It is suggested that episodic information is an essential constituent of olfactory memory and that its function is comparable to that of form and structure in visual and auditory memory systems.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Fetal cells in the maternal blood

Summary In an attempt to stimulate fetal cells in the maternal blood to mitotic division, peripheral blood lymphocytes were cultured from ten primiparous women and six multiparous women. In the case of the ten primiparous women, PWM was used to stimulate lymphocytes in 3- and 7-day cultures made at the 16th, 20th, 24th, and 28th week of gestation. Altogether, 10565 mitoses were analyzed after quinacrine staining of cells from five mothers who each subsequently gave birth to a male infant, and not a single XY mitosis was found.In the case of the multiparous women, lymphocyte cultures, with PHA or LPS as mitogen and MLC, were initiated between the 13th and 20th week of pregnancy. Four of the mothers were pregnant with a male child, and two with a female child. From cultures of each of the four mothers expecting a boy, a total of 9721 mitoses were analyzed after quinacrine staining, and not a single XY mitosis was found. However, one XY cell was found in the culture from one of the two women who delivered a girl. The XY mitosis probably originated from a pregnancy 8 months earlier which terminated in a male infant.In an attempt to culture and obtain good chromosome preparations from small numbers of cells, it was shown that a good mitotic response and good chromosome preparations could be obtained from as few as 6000 lymphocytes.     

Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.     

Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.     

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Differential Roles of the Pseudomonas aeruginosa PA14 rpoN Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.