Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several proteins bind to cdb3, including protein 4.2, a cytoskeletal protein. Three mutations in cdb3 are associated with hereditary spherocytosis (HS) and decreased levels of protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and mutant proteins are dimers. No difference in secondary structure between mutant and wild-type proteins was detected using circular dichroism (CD) analysis. The wild-type and mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3 proteins do not differ to any great extent in structure from the wild-type protein, suggesting that the HS mutations may directly affect protein 4.2 binding.     

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.     

In vivo vitiligo induction and therapy model: double-blind, randomized clinical trial

In this study, we developed an in vivo vitiligo induction model to explore the underlying mechanisms leading to Koebner's phenomenon and to evaluate the efficacy of therapeutic strategies. The model consisted of 12 pigmented test regions on the back of generalized vitiligo patients that were exposed to three Koebner induction methods: cryotherapy, 755 nm laser therapy, and epidermal abrasion. In addition, four cream treatments (pimecrolimus, tacrolimus, steroid and placebo) were randomly applied. Koebnerization was efficiently induced by all three induction methods. In general, cryotherapy was the best method of Koebner induction, followed by 755 nm laser therapy and epidermal abrasion. Reproducible results were obtained, which showed enhanced depigmented surface areas and higher amounts of T lymphocytes in placebo-treated test zones compared to active treated areas. Tacrolimus and local steroids were better inhibitors of Koebner's process (P < 0.05) compared to pimecrolimus. Our in vivo vitiligo induction model is very informative to investigate vitiligo induction and to determine the efficacy of topical treatments in vitiligo. This proof of concept confirms the efficient comparison of head-to-head therapeutic strategies intra-individually in a standardized, specific and better timed way.     

Lipocalin 2 Regulates Inflammation during Pulmonary Mycobacterial Infections

Pulmonary tuberculosis (TB), caused by the intracellular bacteria Mycobacterium tuberculosis, is a worldwide disease that continues to kill more than 1.5 million people every year worldwide. The accumulation of lymphocytes mediates the formation of the tubercle granuloma in the lung and is crucial for host protection against M.tuberculosis infection. However, paradoxically the tubercle granuloma is also the basis for the immunopathology associated with the disease and very little is known about the regulatory mechanisms that constrain the inflammation associated with the granulomas. Lipocalin 2 (Lcn2) is a member of the lipocalin family of proteins and binds to bacterial siderophores thereby sequestering iron required for bacterial growth. Thus far, it is not known whether Lcn2 plays a role in the inflammatory response to mycobacterial pulmonary infections. In the present study, using models of acute and chronic mycobacterial pulmonary infections, we reveal a novel role for Lcn2 in constraining T cell lymphocytic accumulation and inflammation by inhibiting inflammatory chemokines, such as CXCL9. In contrast, Lcn2 promotes neutrophil recruitment during mycobacterial pulmonary infection, by inducing G-CSF and KC in alveolar macrophages. Importantly, despite a common role for Lcn2 in regulating chemokines during mycobacterial pulmonary infections, Lcn2 deficient mice are more susceptible to acute M.bovis BCG, but not low dose M.tuberculosis pulmonary infection.     

Predicting plant responses to mycorrhizae: integrating evolutionary history and plant traits

We assessed whether (1) arbuscular mycorrhizal colonization of roots (RC) and/or plant responses to arbuscular mycorrhizae (MR) vary with plant phylogeny and (2) MR and RC can be more accurately predicted with a phylogenetic predictor relative to a null model and models with plant trait and taxonomic predictors. In a previous study, MR and RC of 95 grassland species were measured. We constructed a phylogeny for these species and found it explained variation in MR and RC. Next, we used multiple regressions to identify the models that most accurately predicted plant MR. Models including either phylogenetic or phenotypic and taxonomic information similarly improved our ability to predict MR relative to a null model. Our study illustrates the complex evolutionary associations among species and constraints of using phylogenetic information, relative to plant traits, to predict how a plant species will interact with AMF.     

Effects of re‐introducing fire to a central Florida sandhill community

Abstract. A southern ridge sandhill site in central Florida, USA, was burned in 1989, 1991, and 1995 after 63 years of fire‐suppression to simulate a pre‐settlement fire regime. Fire changed species abundance and vegetation structure but caused only minimal changes in species turnover and diversity. There was a general trend for an increase in the cover of herbs following fire but this was a statistically significant effect for only one species, Liatris tenuifolia var. tenuifolia. Aristida beyrichiana increased, litter cover and litter depth were significantly reduced, and ground lichens were eliminated in response to burning. Scrub oaks and palmettos in the ground cover and small shrub layers (height ≤ 1 m) either increased or did not respond to burning, reflecting strong post‐fire resprouting. Diversity in the ground cover and small shrub layers were not affected by fire. Scrub oaks and palmettos in the large shrub and overstorey layers (height > 1 m) were reduced in density, basal area, and longest canopy measurements in response to fire. Species diversity also decreased within these layers following fire. Some Pinus elliottii var. densa survived fire, but their density was reduced. All Pinus clausa were eliminated by fire. Periodic burning can suppress the dominance of shrubs (Quercus spp.) while increasing the cover of grasses and herbs in southern ridge sandhill vegetation.     

TRAP: a modelling approach to below-ground carbon allocation in temperate forests

Tree root systems, which play a major role in below-ground carbon (C) dynamics, are one of the key research areas for estimating long-term C cycling in forest ecosystems. In addition to regulating major C fluxes in the present conditions, tree root systems potentially hold numerous controls over forest responses to a changing environment. The predominant contribution of tree root systems to below-ground C dynamics has been given little emphasis in forest models. We developed the TRAP model, i.e. Tree Root Allocation of Photosynthates, to predict the partitioning of photosynthates between the fine and coarse root systems of trees among series of soil layers. TRAP simulates root system responses to soil stress factors affecting root growth. Validation data were obtained from two Belgian experimental forests, one mostly composed of beech (Fagus sylvatica L.) and the other of Scots pine (Pinus sylvestris L.). TRAP accurately predicted (R = 0.88) night-time CO₂ fluxes from the beech forest for a 3-year period. Total fine root biomass of beech was predicted within 6% of measured values, and simulation of fine root distribution among soil layers was accurate. Our simulations suggest that increased soil resistance to root penetration due to reduced soil water content during summer droughts is the major mechanism affecting the distribution of root growth among soil layers of temperate Belgian forests. The simulated annual rate of C input to soil litter due to the fine root turnover of the Scots pine was 207 g C m^–2 yr^–1. The TRAP model predicts that fine root turnover is the single most important source of C to the temperate forest soils of Belgium.     

Growth, photosynthesis and ozone uptake of young beech (Fagus sylvatica L.) in response to different ozone exposures

Beech seedlings (Fagus sylvatica L.) were exposed to episodes of O₃ in environmentally controlled growth chambers during one growing season. Three treatments were applied: charcoal-filtered air, charcoal-filtered air with the addition of 40 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours), and charcoal-filtered air with the addition of 100 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours). The accumulated exposure over a threshold of 40 ppb in the last treatment reached 13,911 ppb h. Throughout the growing season we measured growth as well as photosynthetic properties and related effects to external and calculated internal doses of O₃, using stomatal conductance (g_s) data. Growth, measured as diameter increment and biomass, was not significantly affected by the O₃ treatments. In the 100-ppb treatment, light-saturated CO₂ assimilation rates and chlorophyll content were significantly reduced, and the chlorophyll fluorescence parameter F_v/F_m was significantly reduced at times of high uptake rates and coincided with strong reductions of assimilation rates. O₃ uptake was lowered in the 100-ppb treatment due to reduced g_s. There was serious visible damage by the end of the exposure period in the 100-ppb treatment, while the treatment with 40 ppb O₃ did not seem to cause any significant changes.