Glasshouse vs field experiments: do they yield ecologically similar results for assessing N impacts on peat mosses?

• Peat bogs have accumulated more atmospheric carbon (C) than any other terrestrial ecosystem today. Most of this C is associated with peat moss (Sphagnum) litter. Atmospheric nitrogen (N) deposition can decrease Sphagnum production, compromising the C sequestration capacity of peat bogs. The mechanisms underlying the reduced production are uncertain, necessitating multifactorial experiments. • We investigated whether glasshouse experiments are reliable proxies for field experiments for assessing interactions between N deposition and environment as controls on Sphagnum N concentration and production. We performed a meta-analysis over 115 glasshouse experiments and 107 field experiments. • We found that glasshouse and field experiments gave similar qualitative and quantitative estimates of changes in Sphagnum N concentration in response to N application. However, glasshouse-based estimates of changes in production--even qualitative assessments-- diverged from field experiments owing to a stronger N effect on production response in absence of vascular plants in the glasshouse, and a weaker N effect on production response in presence of vascular plants compared to field experiments. • Thus, although we need glasshouse experiments to study how interacting environmental factors affect the response of Sphagnum to increased N deposition, we need field experiments to properly quantify these effects.

Salivary immune markers are not associated with self-reported childhood maltreatment or psychopathology in adults.

BACKGROUND: Psychological stress has repeatedly been found to be associated with pro-inflammatory markers in blood, and neuro-inflammation may play a role in the development of psychopathology after early life stress. Salivary immune testing is a novel method to non-invasively assess immune functioning. We examined a large range of salivary immune markers in relation to self-reported childhood maltreatment and psychopathology in an adult sample. METHODS: Participants (N = 118, 51% female, mean age = 46.6 yrs, range 22-64) were drawn from a cross-sectional three-generation study, and supplied 2 ml of saliva via passive drool. They reported on childhood maltreatment experiences and on psychopathological symptoms in the last 6 months. Hair cortisol was additionally assessed in a subsample (n = 68). Levels of IL1ß, IL6, IL8, IFNγ, TNFα, tIgE, sIgA, FLCƛ, and FLCƙ were assessed. RESULTS: Linear mixed model analyses showed that several salivary immune markers were associated with age (sIgA and IgE), BMI (sIgA, IL1ß, and IL6), sex (FLCs and IgE), and bad health (IL6, IL8, TNFα). No associations with (anti-inflammatory) medication use or oral health problems were found. Notably, no associations between the immune markers and self-reported childhood maltreatment, psychopathology, or hair cortisol were found. CONCLUSIONS: Salivary immune measures were found to be sensitive to individual differences in age, sex, health and BMI. However. in the current sample there was no indication of inflammation in relation to chronic psychological stress. Larger studies, including participants with higher stress levels, are needed to further examine associations between salivary immune markers and psychological stress.

Climatic modifiers of the response to nitrogen deposition in peat-forming Sphagnum mosses: a meta-analysis.

Peatlands in the northern hemisphere have accumulated more atmospheric carbon (C) during the Holocene than any other terrestrial ecosystem, making peatlands long-term C sinks of global importance. Projected increases in nitrogen (N) deposition and temperature make future accumulation rates uncertain. Here, we assessed the impact of N deposition on peatland C sequestration potential by investigating the effects of experimental N addition on Sphagnum moss. We employed meta-regressions to the results of 107 field experiments, accounting for sampling dependence in the data. We found that high N loading (comprising N application rate, experiment duration, background N deposition) depressed Sphagnum production relative to untreated controls. The interactive effects of presence of competitive vascular plants and high tissue N concentrations indicated intensified biotic interactions and altered nutrient stochiometry as mechanisms underlying the detrimental N effects. Importantly, a higher summer temperature (mean for July) and increased annual precipitation intensified the negative effects of N. The temperature effect was comparable to an experimental application of almost 4 g N m(-2) yr(-1) for each 1°C increase. Our results indicate that current rates of N deposition in a warmer environment will strongly inhibit C sequestration by Sphagnum-dominated vegetation.

Developmental regulation of thymocyte susceptibility to deletion by "self"-peptide.

The specificity of the T cell receptor (TCR) repertoire for foreign peptide bound to self-major histocompatibility complex (MHC) molecules is determined in large part by positive and negative selection processes in the thymus, yet the mechanisms of these selection events remain unknown. Using in vitro organ culture of thymi isolated from mice transgenic for a TCR-alpha/beta specific for cytochrome c peptide bound to I-Ek, we analyzed the developmental timing of negative selection (deletion). On the basis of the experiments described below, we conclude that all CD4+8+ thymocytes, and only CD4+8+ thymocytes, are susceptible to negative selection mediated by the cytochrome c peptide antigen. First, we found that deletion of thymocytes resulting from addition of the cytochrome c peptide to the thymic organ cultures can occur at the earliest stage of TCR, CD4, and CD8 coexpression. Second, we found that CD4+8+ thymocytes isolated from positively selecting or nonselecting MHC haplotypes were equally efficiently deleted in vitro, suggesting that positive selection is not a prerequisite for deletion. Third, we examined the effects of TCR/ligand avidity on the developmental timing of deletion by varying the concentration of cytochrome c peptide added to the organ cultures. We detected deletion only at the CD4+8+ stage: intermediate concentrations of peptide that resulted in partial deletion of CD4+8+ cells did not eliminate the appearance of mature CD4+8- cells. Finally, we found that CD4+8- thymocytes were resistant to deletion as well as activation by peptide antigen added to the intact organ cultures. Nevertheless, the CD4+8- thymocytes isolated from the peptide-treated organ cultures responded vigorously to peptide presented by spleen cells in vitro. Thus, the T cells were tolerant of (but not anergized by) self-antigen encountered in thymic organ culture. Together, these results indicate that thymocytes susceptible to negative selection are not developmentally distinct from those susceptible to positive selection, and further, that the thymic microenvironment plays a role in regulating the outcome of TCR/ligand interactions.

Peripheral expression of Jak3 is required to maintain T lymphocyte function.

The Jak family tyrosine kinase, Jak3, is involved in signaling through cytokine receptors that utilize the common gamma chain (gammac), such as those for IL-2, IL-4, IL-7, IL-9, and IL-15. Recent studies of Jak3-deficient mice and humans have demonstrated that Jak3 plays a critical role in B and T lymphocyte maturation and function. The T lymphocyte defects in Jak3-deficient mice include a small thymus, a decrease in peripheral CD8+ cells, an increase in the surface expression of activation markers, and a severe reduction in proliferative and cytokine secretion responses to mitogenic stimuli. To determine whether the peripheral T lymphocyte defects result from aberrant maturation in the thymus or from the absence of Jak3 protein in peripheral T cells, we generated reconstituted mice that express normal levels of Jak3 protein in the thymus but lose Jak3 expression in peripheral T cells. Jak3 expression in the thymus restores normal T cell development, including CD8+, gammadelta, and natural killer cells. However, the loss of Jak3 protein in peripheral T cells leads to the Jak3-/- phenotype, demonstrating that Jak3 is constitutively required to maintain T cell function.

T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells.

Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk-/- T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk-/- T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C gamma1 tyrosine phosphorylation are substantially reduced in Itk-/- T cells. In contrast, TCR-zeta and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.

Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility.

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)alpha-/- murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-gamma locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Ralpha to rearrangement of the TCR-gamma locus requires the gammac receptor chain and the gammac-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-gamma locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Ralpha-/- thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Ralpha-/- thymocytes. Instead, the TCR-gamma locus was shown to be methylated in IL-7Ralpha-/- thymocytes. Treatment of IL-7Ralpha-/- precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-gamma gene rearrangement. This data supports the model that IL-7R promotes TCR-gamma gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.

Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3.

Biochemical studies of signaling mediated by many cytokine and growth factor receptors have implicated members of the Jak family of tyrosine kinases in these pathways. Specifically, Jak3 has been shown to be associated with the interleukin-2 (IL-2) receptor gamma chain, a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mice lacking Jak3 showed a severe block in B cell development at the pre-B stage in the bone marrow. In contrast, although the thymuses of these mice were small, T cell maturation progressed relatively normally. In response to mitogenic signals, peripheral T cells in Jak3-deficient mice did not proliferate and secreted small amounts of IL-2. These data demonstrate that Jak3 is critical for the progression of B cell development in the bone marrow and for the functional competence of mature T cells.

Reduced nitrogen has a greater effect than oxidised nitrogen on dry heathland vegetation.

We investigated the effects of different ratios of reduced (NH4+) versus oxidised (NO3(-)) nitrogen in deposition on heathland and species-rich grassland vegetation at high nitrogen deposition levels in large mesocosms filled with nutrient-poor soils to which different NH4+/NO3(-) ratios were applied. The response of the forbs, Antennaria dioica, Arnica montana, Gentiana pneumonanthe, Thymus serpyllum, the grasses Danthonia decumbens, Deschampsia flexuosa, Nardus stricta and the shrub Calluna vulgaris was recorded. The forb A. dioica and the grass D.decumbens preferred low NH4+/NO3(-) ratios and were characterised by a negative correlation between NH4+/NO3(-) ratios and biomass and survival, whereas the grasses N. stricta and D. flexuosa showed no correlation with NH4+/NO3(-) ratios. Lime addition eliminated the negative effects of high NH4+ concentrations in deposition for A. dioica and the grass D. decumbens. The implications of these findings for heathland vegetations are discussed.

Molecular determinants of TCR expression and selection.

The process of T cell development in the thymus is tightly regulated, being dependent on the integration of signals required for thymocyte maturation and survival. Rearrangements, expression and signaling of TCR genes play an indispensable role in this developmental program. Recent advances have provided insights into the molecular mechanisms that regulate TCR repertoire formation at the level of alphabeta versus gammadelta T cell fate and CD4(+) versus CD8(+) lineage determination.

The role of Jak3 in lymphoid development, activation, and signaling.

Mutations in a number of lymphoid signaling molecules lead to immunodeficiencies in mice and humans. Among these, one very pleiotropic syndrome results from deficiencies in an array of cytokine signaling pathways utilizing a cytokine receptor common gamma chain, gammac, and the tyrosine kinase Jak3. Recent advances in our understanding of the role of gammac and Jak3 in lymphocyte development and function highlight the importance of cytokine receptor signaling pathways in regulating lymphoid homeostasis and responsiveness.

The role of cytokine receptor signaling in lymphocyte development.

Cytokine receptor signaling plays an essential role in the early stages of lymphocyte development. Signals through various cytokine receptors - such as c-kit, flt3/flk2, CXCR4, the IL-7 receptor and the IL-15 receptor - are known to promote the expansion and survival of uncommitted progenitor cells as well as their migration to the appropriate microenvironment and subsequent differentiation into B, T or natural killer cells. The recent generation of mice deficient in one or more of these signaling pathways has revealed which cytokines play unique and/or redundant roles in each of these lymphocyte lineages during this developmental process.

Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene.

We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number.

Expression of T-cell receptor alpha-chain genes in transgenic mice.

To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).

A new member of the Eph family of receptors that lacks protein tyrosine kinase activity.

Using a PCR-based screen to identify tyrosine kinases involved in T cell development, we have cloned a new member of the Eph-family of receptor tyrosine kinases (Mep, for murine eph-family protein). At the amino acid level Mep is 60% identical to the chicken embryonic kinase Cek9. Sequence analysis indicates that the predicted extracellular portion of Mep bears an Ig-like domain, a cysteine-rich region, and sequences homologous to fibronectin type III. The transmembrane region of Mep is followed by a kinase domain. Surprisingly, this kinase domain carries amino acid substitutions in the highly conserved consensus motifs found in all protein tyrosine kinases and known to be crucial for kinase activity. We demonstrate that a bacterial fusion protein of the Mep kinase domain does not have protein tyrosine kinase activity. Analysis of Mep mRNA levels in a variety of mouse tissues shows that Mep is highly expressed in thymus and brain. We have also isolated two additional Mep cDNA clones from thymocytes which are predicted to encode secreted forms of the Mep extracellular domain; mRNAs encoding these secreted isoforms are also expressed in mouse brain.

Single-family versus multi-family introductions.

The early success of plant introduction as a function of source material and habitat quality was tested in Arnica montana to determine whether different introduction strategies could affect plant viability of the introduced population. Plants originating from related (single-family introductions) and unrelated (multi-family introductions) individuals were introduced into undisturbed sites and into sites which were limed, turf cut, or both turf cut and limed. For four consecutive years, we analysed plant performance by measuring survival time, growth, and reproductive capacity. Introduction success was found to be strongly affected by habitat quality. Turf cutting in combination with additional liming significantly increased reproductive capacity and stimulated early flowering. To restore eutrophic and acidified soil conditions, turf cutting with additional liming prior to introduction is recommended. Furthermore, a significant effect of multi-family introductions was observed. Multi-family introductions showed higher introduction success compared to single-family introductions. Although the long-term effects of multi-family introductions will emerge after several generations, the preliminary results suggest multi-family introductions as the most successful introduction strategy.

T cell development and activation in Jak3-deficient mice.

Jak3, a member of the Janus family of tyrosine kinases, participates in signaling through cytokine receptors that contain the common gamma-chain, including the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15. Jak3- and gamma c-deficient mice have pleiotropic defects that can be attributed to their inability to respond to multiple specific cytokines. A great deal of recent work has focused on the T cell defects in these mutant mice. Specifically, Jak3- and gamma c-deficient mice have small thymuses revealing a defect in T cell development, and in addition, have functionally unresponsive peripheral T cells with an activated/memory cell phenotype. The thymic defect in these mutant mice strongly resembles that seen in IL-7 and IL-7 receptor knockout mice, suggesting that the lack of IL-7 receptor signaling accounts for this defect in Jak3-/- and gamma c- mice. To characterize this defect further, we have examined the earliest stages of T cell development in adult and fetal Jak3-/- thymuses. These studies identify two discrete developmental defects at the CD4-CD8- stage of T cell maturation. Analyses of peripheral T cells in Jak3-/- and gamma c- mice have also revealed a number of abnormalities. All of the T cells in these mutant mice have an activated phenotype and a large fraction of them are proliferating in vivo. In addition, Jak3-/- and gamma c- T cells are more prone to undergo apoptosis than wild-type T cells. Together, these features account for the decreased IL-2 secretion by in vitro-stimulated Jak3-/- T cells. Overall, many of the lymphoid defects of Jak3- and gamma c-deficient mice can be accounted for by the lack of IL-7R and IL-2R signaling; however, other cytokine systems must also be involved in maintaining peripheral T cell homeostasis.

Genome sizes of 227 accessions of Gagea (Liliaceae) discriminate between the species from the Netherlands and reveal new ploidies in Gagea.

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Gagea (Liliaceae), mainly from the Netherlands. The basic chromosome number for Gagea is x = 12. The inferred ploidy in the Dutch and German accessions varies from diploid to decaploid. Consequently there is a large range of genome sizes (DNA 2C-values) from 14.9 to 75.1 pg. Genome sizes are evaluated here in combination with the results of morphological observations. Five species and the hybrid G. × megapolitana are reported. Apart from 14 diploid G. villosa, six plants of G. villosa with an inferred tetraploidy were found. For the 186 Dutch accessions investigated 85 turned out to be the largely sterile G. pratensis (inferred to be pentaploid). Inferred tetraploid and hexaploid G. pratensis were found in 30 and 20 localities, respectively. In one locality an inferred decaploid (10×) plant was found that could represent a doubled pentaploid G. pratensis. An inferred decaploid G. pratensis was never reported before. The genome size of Gagea × megapolitana from Germany fitted with its origin as a cross between the two hexaploids G. pratensis and G. lutea. Gagea spathacea from the Netherlands was inferred to be nonaploid as was recorded from plants across Europe. The aim of the study was to use flow cytometry as a tool to elucidate the taxonomic position of the Dutch Gagea.

Genomic structure and promoter region of the murine Janus-family tyrosine kinase, Jak3.

Genomic DNA sequences encoding the murine Janus family tyrosine kinase Jak3 were isolated to determine the intron-exon structure of the gene and to investigate the phylogeny of Jak-family kinases. The murine Jak3 gene comprises approximately 15 kbp of genomic DNA and consists of 23 exons. The organization of sequences encoding the pseudo-kinase domain of Jak3 is similar to the intron-exon structure encoding catalytic domains of Src-family tyrosine kinases, whereas the pattern of introns-exons encoding the Jak3 kinase domain shows no structural similarity to that of other tyrosine kinase genes. Genomic analysis further indicates that alternative splicing gives rise to different forms of the murine Jak3 mRNA encoding different isoforms of the Jak3 protein. Analysis of Jak3 intron-exon structure also suggests that a mutation in the human JAK3 gene responsible for a severe combined immune deficiency (SCID) phenotype results from aberrant splicing of the JAK3 transcript. Finally, potential regulatory sequences in the upstream region of the murine Jak3 gene were analyzed and are discussed in relation to the known expression pattern of Jak3.

Chromophore-assisted laser inactivation of subunits of the T-cell receptor in living cells is spatially restricted.

Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.