Surface Phosphatase Research Articles

Orientation-dependent CD45 inhibition with viral and engineered ligands.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

Wheat Straw Burial Enhances the Root Physiology, Productivity, and Water Utilization Efficiency of Rice under Alternative Wetting and Drying Irrigation

This study evaluated whether the straw burial and alternative wetting and drying (AWD) irrigation could improve the root activity, yield, and water utilization efficiency (WUE) of rice. Accordingly, we conducted a field experiment with three straw burial levels, i.e., with no straw burial (NSB), low straw burial 300 kg.ha−1 (LSB), and dense straw burial 800 kg.ha−1 (DSB), and three irrigation regimes, i.e., alternate wetting/moderate drying (AWMD), alternate wetting/severe drying (AWSD), and alternate wetting/critical drying (AWCD). Results showed that straw burial improved the root activity, rice yield, and WUE under AWD regimes. The combination AWMD×DSB resulted in the greatest values of total dry mass (1764.7 g/m2) and water use (853.1 mm). Conversely, the treatment AWCD × NSB led to the lowest values of total biomass (583.3 g/m2) and water use (321.8 mm). Root dry weight density (1.11 g cm−3) and root active absorption area (31.6 m2 plant−1) were higher in the treatment AWMD × DSB than root dry weight density (0.41 g cm−3) and root active absorption area (21.2 m2 plant−1) were in the treatment AWCD×NSB. The former combined treatment increased root oxidation ability (55.5 mg g−1 FWh−1), the root surface phosphatase activity (1.67 mg g−1 FWh−1) and nitrate reductase activity of root (14.4 μg g−1 h−1) while the latter considerably reduced the values of root oxidation ability (21.4 mg g−1 FWh−1), the root surface phosphatase activity (0.87 mg g−1 FWh−1) and nitrate reductase activity of root (5.8 μg g−1 h−1). The following conclusions can be drawn with regard to water use and biomass yield. (i) The reduction in water consumption was greater than the reduction in yield in the case of AWSD. (ii) The decline in water consumption was less than the decline in biomass yield in the case of AWCD. (iii) The increase in in water consumption was greater than the increase in biomass yield in the case of AWMD. Therefore, the indicators of WUE were recorded in the following order: AWSD > AWMD > AWCD. This study recommends AWD irrigation to improve the root growth traits that contribute to the greater biomass yield of rice. It also suggests that farmers should implement AWD irrigation after leaving wheat straw residues in the field, and followed by deep tillage, to mitigate the negative effect of drought stress caused by AWD irrigation, preserving plant growth without large biomass losses, and thus, addressing the constrains of straw residues and sustaining rice production under limited freshwater resources.

Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism.

Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45–NKG2A and CD45–Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.

Impact of alternative wetting and soil drying and soil clay content on the morphological and physiological traits of rice roots and their relationships to yield and nutrient use-efficiency

Rice is threatened by the inefficient use of water and fertilizer. However, understanding the effects of water regimes and soil clay contents on morphology and physiology of plant roots and their relations to yield and nutrient use-efficiency is essential to improve rice productivity. In this regard, three experimental soils were conducted with three soil clay content levels, namely, light clay (S LC), medium clay (S MC), and heavy clay (S HC), and three irrigation regimes, namely, alternate wetting and 0% soil drying (AWSD0%), alternate wetting and 10% soil drying (AWSD10%), alternate wetting and 30% soil drying (AWSD30%) over July-October in 2018. The experiment was a randomized complete block design in a factorial arrangement with three replications. The results showed that responses of roots and shoots and the nitrogen (N), phosphorus (P) and potassium (K) uptake of rice were significantly affected by the water regimes and soil clay contents. Under the same water regime, root morphology and physiology, biomass production and NPK utilization of rice were higher in high clay soil than light clay soil. Under the same soil clay content, AWSD0% regime was the optimal water management practice for growing rice, providing the highest grain yield. Whereas, AWSD30% regime was improper water management option for rice, offering the lowest grain yield. The combination, AWSD0%×S HC, resulted in the greatest values of the apparent recovery efficiency (ARE) of NPK applied, at AREN (43.4%), AREP (20.4%) and AREK (67.2%), as well as the highest values of partial factor productivity (PFP) of NPK, applied, at PFPN (19.7, g g−1), PFPP (31.9, g g−1) and PFPK (41.1, g g−1) respectively. Conversely, the treatment AWSD30%×S LC resulted in the lowest values of ARE of NPK applied at AREN (8.3%), AREP (3.0%), and AREK (6.7%), as well as the lowest values of PFP of NPK, applied at PFPN (7.0, g g−1), PFPP (11.9, g g−1) and PFPK (12.2, g g−1) respectively. Root length density, root weight density, root active absorption area, root oxidation ability, and root surface phosphatase were higher in AWSD0%×S HC than that in AWSD70% × S LC. While the former combination increased NPK contents in the rhizosphere, the latter their reduced availability due to the increment in pH and redox potential of the soil. The results are important for rice water management option in the paddy clay soil. The results also suggest that the adoption of alternate wetting and soil drying regimes should guarantee morphological and physiological improvements in root traits, by which contributes to greater biomass production, larger NPK acquisition and, thus better NPK utilization of rice.

Cascade enzymes within self-assembled hybrid nanogel mimicked neutrophil lysosomes for singlet oxygen elevated cancer therapy

As the first line of innate immune cells to migrate towards tumour tissue, neutrophils, can immediately kill abnormal cells and activate long-term specific adaptive immune responses. Therefore, the enzymes mediated elevation of reactive oxygen species (ROS) bioinspired by neutrophils can be a promising strategy in cancer immunotherapy. Here, we design a core-shell supramolecular hybrid nanogel via the surface phosphatase triggered self-assembly of oligopeptides around iron oxide nanoparticles to simulate productive neutrophil lysosomes. The cascade reaction of superoxide dismutase (SOD) and chloroperoxidase (CPO) within the bioinspired nanogel can convert ROS in tumour tissue to hypochlorous acid (HOCl) and the subsequent singlet oxygen (1O2) species. Studies on both cells and animals demonstrate successful 1O2-mediated cell/tumour proliferation inhibition, making this enzyme therapy capable for treating tumours without external energy activation.

Abstract B59: Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery

Abstract Adoptive cell transfer (ACT) with autologous tumor-reactive T-cells has recently shown great promise in the treatment of leukemia in clinical trials. Effective control of solid tumors, remain challenging. Glioblatoma multiforme (GM) is a lethal disease in need of clinically effective therapies. Ninety-seven percent of tumors in the ‘classical’ subtype carry extra copies of the epidermal growth factor receptor (EGFR) gene, and most have higher than normal expression of EGFR. In the present study we utilize a novel carrier-free delivery strategy to specifically support a EGFR directed chimeric antigen receptor (CAR) T-cell with recombinant Interleukin-15 super agonist (IL15-Sa), in a heterotopic mouse model of GM. CAR-T cell design: The CAR-T cell was designed based on the heavy and light chains of cetuximab to form a single-chain variable fragment, which was fused to a portion of the extracellular and transmembrane domains of human CD8 alpha, followed by the intracellular domains of 4-1BB and CD3 zeta. The bicistronic vector also encoded truncated human CD19 as a selectable marker. The plasmid coding hu EGFRscFv-BBz-CAR was synthesized and lentivirus packaging was made. Isolated T cells were derived from leukapheresis products and stimulated with Dynabeads Human T Activator CD3/CD28. T cell were cultured and were transduced and or left untransduced one day following bead stimulation, and then T cells were expanded for 10 days and cryopreserved. Surface expression of the CAR was confirmed and quantified with biotinylated human EGFR protein and by staining for CD19. CAR-T cells phenotype was identified. Coupling of NGs to T cells: anti CD45 antibody (aCD45) against highly expressed cell surface phosphatase CD45 was used as an anchor to stably link human IL-15Sa as a drug cargo/nanogel (NG) to the T cell surface. The aCD45/IL-15-Sa-NG was added to CAR T cells. In vivo therapy study using human Chimeric Antigen Receptor (CAR)-T cells: Luciferase-expressing U-87 MG human glioblastoma cells (1.0 × 106) were injected s.c. into NSG mice on Day 0. Animals received i.v. adoptive transfer of activated CAR-T cells (2.6×106 total T-cells -38% transduction equating to 106 CAR-T-cells)) alone or with surface coupled NGs on Day 7. In other groups, free IL-15Sa was injected i.v. after ACT at equivalent dose. Tumor area and body weight were measured every two days. Mice were also imaged for bioluminescence to monitor the tumor growth and were euthanized when body weight loss was beyond 20%, or tumor area reached 150 mm2 or the animal had become moribund. Findings: CAR T-cells backpacked with IL-15Sa-NGs were compared to CAR T-cells alone or T-cells supplemented with an equivalent systemic dose of free IL-15Sa. NG-backpacked CAR T-cells eradicated tumors in 80% of animals. By contrast, transfer of CAR-T cells alone had a small impact on tumor growth and survival, which did not reach statistical significance; responses were marginally improved by the addition of free IL-15Sa. We have demonstrated NG delivery of cytokines has the potential to enhance CAR T-cell therapy in a mice model of GM. This strategy should be broadly applicable in ACT strategies employing natural, TCR-engineered, or CAR T-cell therapies, and illustrates a general approach to couple the action of a synthetic drug delivery carrier to the function of therapeutic cells. Citation Format: Ana Castano, Li Tang, Sagar Kudchodkar, Irvine Darrell, Marcela Maus. Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B59.

Small size of membrane-tethered ligands regulates kinase-phosphatase segregation in γδ TCR triggering.

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not undergo canonical thymic selection, and consequently are not restricted by pMHC antigens or the co-receptors CD4/8. Some of these unconventional T cells recognize invariant membrane-tethered ligands, and their rapid activation and recruitment to sites of inflammation serves as a ‘first line of defense’ against incipient infection. The substantial structural and stoichiometric similarities between αβ and γδ TCRs prompted us to investigate whether γδ TCR ligand-recognition and triggering shared basic physicochemical constraints with conventional αβ TCRs. We chose as a model system the stress-inducible murine surface protein T22 that is recognized by the G8 γδ TCR - for which detailed biophysical and structural information is available. Since T22 is similar in size to pMHC molecules, we tested whether ‘size-based’ kinase-phosphatase segregation, following G8 engagement by T22, controls initiation of TCR signaling. In our ongoing studies, we have established that the small axial size of T22 is critical for G8 triggering. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 responses. Using optical and electron microscopy we show that elongated T22/G8 complexes increases access of inhibitory phosphatases to sites of TCR engagement at cell-cell contacts. We are testing whether truncation of the ectodomain of the large cell surface phosphatases CD45/CD148, which are normally sterically segregated from engaged TCR, affects triggering. Finally, we are adapting these strategies, to investigate mechanisms of triggering by γδ T cells that recognize small-molecule organic compounds.

A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions

The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

Surface phosphatase inRhinocladiella aquaspersa: biochemical properties and its involvement with adhesion

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

Combined effects of nitrogen enrichment, sulphur pollution and climate change on fen meadow vegetation N:P stoichiometry and biomass

Nitrogen (N) and sulphur (S) deposition, as well as altered soil moisture dynamics due to climate change can have large effects on fen meadow biogeochemistry and vegetation. Their combined effects may differ strongly from their separate effects, since each process affects different nutrients through different mechanisms. However, the impacts of these environmental problems are rarely studied in combination. We therefore investigated the separate and interactive effects of current levels of N- and S-deposition and changes in soil moisture dynamics on fen meadow vegetation. We focused on vegetation biomass and N:P stoichiometry, including access to soil P through root surface phosphatase activity, in a 3-year factorial addition experiment in an N-limited rich fen meadow in the Biebrza valley in Poland. We applied 29.5 kg N ha−1 year−1 and 32.1 kg S ha−1 year−1, which correspond to current deposition levels in Western Europe. Changes in soil moisture dynamics due to climate change were mimicked by amplified drying of the soil in summer. This level of N-deposition had limited effects on plant biomass production in this rich fen, despite low foliar N:P ratios that suggest N limitation. This level of S-deposition, however, resulted in decreased vegetation P-uptake and biomass. We also showed that increased summer drought resulted in considerable increases in vegetation biomass. We found no interactive effects on vegetation biomass or N:P stoichiometry, possibly as a result of the limited main effects of the separate processes.

Phosphorus and Nitrogen Fertilization Effect on Phosphorus Uptake and Phosphatase Activity in Ryegrass and Tall Fescue Grown in a Chilean Andisol

A series of short-term experiments were carried out to assess the effect of phosphorus (P) and nitrogen (ammonium [NH4+-N] or nitrate [NO3−-N]) fertilization on P uptake and phosphatase activity in ryegrass and tall fescue cultivated under greenhouse conditions. Ryegrass or tall fescue plants were grown in an acidic Andisol in the presence or absence of P and increasing doses of NO3−-N or NH4+-N fertilizers. At the end of the experiment, soil phosphatase activity (P-aseRhiz), pH, and Olsen-P were determined in the rhizosphere soil. Plant biomass, P uptake, and root surface phosphatase (P-aseRoot) were also assayed for both plant species. Furthermore, soil incubation experiments at increasing doses of P, NO3−-N, or NH4+-N were performed to evaluate the fertilizer effect on soil phosphatase activity (P-aseBulk) and microbial biomass carbon in the bulk soil. In the absence of plants, P-aseBulk was inhibited and microbial biomass carbon was raised at increasing P supply levels. In the greenhouse experiments, P uptake by tall fescue was about 67% higher than that of ryegrass at low soil P availability, which suggests that tall fescue was less sensitive to P deficiency than ryegrass. For both plant species, P-aseRhiz did not vary as a consequence of P addition. On the other hand, fertilization with the highest NH4+-N dose strongly decreased soil pH and shoot P content, as well as it increased P-aseRoot activity. This fact denotes that P-aseRoot behaved as a strategic response parameter to P stress with insufficient impact on plant P nutrition in both plant species.

Increased N affects P uptake of eight grassland species: the role of root surface phosphatase activity

Increased N deposition may change species composition in grassland communities by shifting them to P limitation. Interspecifi c diff erences in P uptake traits might be a crucial yet poorly understood factor in determining the N eff ects. To test the eff ects of increased N supply (relative to P), we conducted two greenhouse fertilization experiments with eight species from two functional groups (grasses, herbs), including those common in P and N limited grasslands. We investigated plant growth and P uptake from two P sources, orthophosphate and not-readily available P (bound-P), under diff erent N supply levels. Furthermore, to test if the N eff ects on P uptake was due to N availability alone or altered N:P ratio, we examined several uptake traits (root-surface phosphatase activity, specifi c root length (SRL), root mass ratio (RMR)) under varying N:P supply ratios. Only a few species ( M. caerulea , A. capillaris , S. pratensis ) could take up a similar amount of P from bound-P to that from orthophosphate. Th ese species had neither higher SRL, RMR, phosphatase activity per unit root (Pase root ), nor higher total phosphatase activity (Pase tot : Pase root times root mass), but higher relative phosphatase activity (Pase rel : Pase tot divided by biomass) than other species. Th e species common from P-limited grasslands had high Pase rel . P uptake from bound-P was positively correlated with Pase tot for grasses. High N supply stimulated phosphatase activity but decreased RMR and SRL, resulting in no increase in P uptake from bound-P. Pase root was infl uenced by N:P supply ratio, rather than by only N or P level, whereas SRL and RMR was not dominantly infl uenced by N:P ratio. We conclude that increased N stimulates phosphatase activity via N:P stoichiometry eff ects, which potentially increases plant P uptake in a species-specifi c way. N deposition, therefore, may alter plant community structure not only by enhancing productivity, but also by favouring species with traits that enable them to persist better under P limited conditions.

Phosphatase activities of cyanobacteria as indicators of nutrient status in a Pyrenees river

There is increasing evidence that fluvial systems are influenced by anthropogenic factors, and disturbance due to pollution and other human interference gives rise to specific problems. It is now imperative that we develop and apply novel and effective ways which allow us to monitor water quality. The present study was planned as part of a programme to develop biological monitoring methods to assess nutrient characteristics of upland calcareous streams and rivers. Phototrophs respond to environmental changes over a period of time, so organisms sampled at one time can potentially provide almost as much information about nutrients in the water as a number of individual chemical measurements. Phosphatase activity is often a good indicator of phosphorus limitation, and field materials could be used to study changes in nutrients dynamics. The calcareous River Muga, north-east Spain, at a site 10 km downstream from its source in the Pyrenees, was therefore chosen for the present study of surface phosphatase activities of the main cyanobacterial communities at different seasons to assess P limitation and establish the suitability of this method for use in monitoring catchment processes. Here we report seasonal changes of phosphatase activity in field populations of Schizothrix coriacea, Rivularia biasolettiana, Tolypothrix distorta var. penicillata and Nostoc verrucosum. All four cyanobacteria showed marked surface phosphomonoesterase and phosphodiesterase activity on each sampling period. Michaelis-Menten kinetic studies showed similar K m values for the four species suggesting similar affinity for organic P substrates. Light had no effect on phosphatase activities, indicating that there is no need to consider this factor in short-term field assays. However, there was an increase in phosphomonoesterase activity of Rivularia with rise in temperature over the range 10–35°C, which suggests adaptation to the frequent temperature changes in nature. Phosphorus limitation seems the main chemical factor influencing phototrophs in R. Muga. Combined observations on macroscopically visible phototrophs with assays of surface phosphatase activity provide a valuable means of assessing long-term changes in a catchment.

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates

This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Requirement for CD45 in fine-tuning mast cell responses mediated by different ligand–receptor systems

The receptor-like protein tyrosine phosphatase CD45, the most abundant cell surface phosphatase on all nucleated hemopoietic cells, is a critical regulator of the activation status of Src family kinases (SFKs). To study the impact of CD45 on mast cell function, we compare bone marrow-derived mast cells (BMMCs) from CD45-deficient mice and from mice expressing an activating point mutation (E613R) in the juxtamembrane wedge of CD45. In response to Ag-triggered FcεR1-mediated activation, CD45-deficient BMMCs exhibit increased inhibitory Lyn phosphorylation and drastically reduced effector functions (degranulation and cytokine secretion). In contrast, CD45 E613R BMMCs show stronger effector functions after Ag-triggering than wild-type (WT) BMMCs. Despite these dichotomous phenotypes, phosphorylation of the inhibitory tyrosine in the SFK Lyn of CD45 E613R BMMCs is comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fyn-regulated phosphatidylinositol-3-kinase pathway. Interestingly, depending on the receptor system addressed, CD45-deficient and CD45 E613R BMMCs show uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or SF is enhanced compared to WT BMMCs. Together, the data indicate that CD45 plays a complex and essential role in fine-tuning mast cell responses mediated by different ligand–receptor systems.

Aquatic plant nutrients, moss phosphatase activities and tissue composition in four upland streams in northern England

Summary A study was made of the water chemistry, tissue nutrients and surface phosphatase activities of the 2-cm apices of three mosses in four upland streams in northern England, UK. This was part of a project to optimize methods for assessing nutrient fractions in environments with highly variable water chemistry. Aqueous N and P fractions showed the greatest variability followed by moss phosphatase activities, with nutrient composition of the shoot apices the least variable. There was no consistent pattern as to which aqueous N or P fraction was the most variable. The ratio between total inorganic N and total filtrable P ranged over three orders of magnitude in some streams. The interrelations between tissue N and P concentrations, tissue N:P ratio, phosphatase activities and aqueous variables showed: (1) Significant +ve relationship between tissue N and aqueous NO 3 –N in some populations, but not between tissue P and aqueous P concentration; (2) Significant +ve relationships between phosphatase activities and aqueous organic N, but none with aqueous organic P; (3) Significant +ve relationships between phosphodiesterase:phosphomonoesterase activities and aqueous organic N; (4) Significant −ve relationships between phosphatase activities and tissue P concentration; (5) Significant +ve relationships between phosphatase activities and tissue N:P. Both types of biological measurement are valuable for monitoring ambient nutrients in upland streams. Neither is clearly better than the other, so both should be included in surveys.

Seasonal changes in the surface phosphatase kinetics of aquatic mosses in northern England

The widespread occurrence of surface phosphatase activity in aquatic mosses, presumably synthesized to utilize organic phosphates in the environment, suggests the likelihood of strategies to optimize this process. Here we report seasonal changes in the kinetics of surface phosphatase in six moss populations, representing four species (Fontinalis antipyretica, Fontinalis squamosa, Rhynchostegium riparioides and Warnstorfia fluitans) from N.E. England. Kinetic parameters of shoots assayed within a few hours of sampling from the streams showed a summer/autumn increase in Km and Vmax corresponding to seasonal decrease in phosphate supply. The detection of phosphomonoesterases and phosphodiesterases with both low and high substrate affinities in all the populations studied confirms the widespread existence of this phenomenon in mosses. Similar two-phase kinetics were found for clonal, axenic isolates of two of the species (Rhynchostegium and Warnstorfia), indicating that any influence of epiphytes on the results with stream material was negligible. The seasonal changes in the kinetic parameters indicate that surface phosphatase activity is a dynamic response to seasonal changes in nutrient supply and/or requirement.

Long-term nitrogen deposition increases phosphorus limitation of bryophytes in an ombrotrophic bog

Here we investigate the effect of 4 years simulated atmospheric deposition of ammonium (NH4) and nitrate (NO3), applied alone or in combination with phosphorus and potassium (PK), on the surface phosphatase activities and nutrient acquisition behaviour of two species of moss (Sphagnumcapillifolium and Hypnum jutlandicum) from an ombrotrophic peatland. Phosphatase activity was significantly enhanced by both the NH4 and NO3 treatments, particularly for Sphagnum, but the activity decreased when exposed to additions of PK. Regression analysis revealed that phosphatase activity on Sphagnum was positively related with tissue N and negatively related to tissue P concentrations. For Hypnum, a negative relationship between shoot P concentration and phosphatase activity was observed. Using a 32P tracer, mosses removed from plots receiving PK in combination with NH4 maintained their affinity for increased phosphorus uptake. These findings suggest that enhanced nutrient supply, even at modest doses, significantly alter the nutrient recycling behaviour of bryophytes.

Phosphatase activities of the aquatic moss Warnstorfia fluitans (Hedw.) Loeske from an acidic stream in North-East England

A study was made of the aquatic environment, tissue nutrient composition and surface phosphatase activities of the aquatic moss Warnstorfia fluitans in Brandon Pithouse Stream, a small acidic stream in N-E England. The water, which originates from an underground spring, had been pH 2.6 for at least 30 years, but about 3.9 during the present study. The moss was by far the most abundant phototroph during all this period. Seasonal changes in aqueous nitrogen and phosphorus fractions were measured over a 2-year period near the source. Most of the filtrable N and P were at times organic, but the very high N:P ratio (even if organic N is excluded) suggests that only organic phosphate is likely to be important for the moss. There was a high peak in organic phosphate in late spring in both study years. Surface phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities were highly correlated in the field and in axenic culture, though there were some differences in response to environmental factors. Axenic material showed higher PMEase and PDEase activities when grown with organic P than with inorganic P. Although the data suggest that internal P content is an important factor influencing phosphatase activities, PDEase activity was especially marked when the moss was grown with the diester, DNA, as P source, indicating that at least one of its surface phosphatases can also respond directly to the environment.

An ectophosphatase activity inCryptococcus neoformans

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.