Neuroprotective immunity: T cell-derived glutamate endows astrocytes with a neuroprotective phenotype

A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 +/- 14 to 9 +/- 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid beta-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.     

Integrated management systems and N fertilization: effect on soil organic matter in rice-rapeseed rotation

Aims

Understanding the effects of long-term crop management on soil organic matter (SOM) is necessary to improve the soil quality and sustainability of agroecosystems.

Method

The present 7-year long-term field experiment was conducted to evaluate the effect of integrated management systems and N fertilization on SOM fractions and carbon management index (CMI). Two integrated soil-crop system management (ISSM-1 and ISSM-2, combined with improved cultivation pattern, water management and no-tillage) were compared with a traditional farming system at three nitrogen (N) fertilization rates (0, 150 and 225 kg N ha^?1).

Results

Management systems had greater effects on SOM and its fractions than did N fertilization. Compared with traditional farming practice, the integrated management systems increased soil organic carbon (SOC) by 13 % and total nitrogen (TN) by 10 % (averaged over N levels) after 7 years. Integrated management systems were more effective in increasing labile SOM fractions and CMI as compared to traditional farming practice. SOC, TN and dissolved organic matter in nitrogen increased with N fertilization rates. Nonetheless, N addition decreased other labile fractions: particulate organic matter, dissolved organic matter in carbon, microbial biomass nitrogen and potassium permanganate-oxidizable carbon.

Conclusions

We conclude that integrated management systems increased total SOM, labile fractions and CMI, effectively improved soil quality in rice-rapeseed rotations. Appropriate N fertilization (N150) resulted in higher SOC and TN. Though N application increased dissolved organic matter in nitrogen, it was prone to decrease most of the other labile SOM fractions, especially under higher N rate (N250), implying the decline of SOM quality.     

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.     

Fertile asymmetric somatic hybrids between Lycopersicon esculentum Mill. and Lycopersicon peruvianum var. dentatum Dun.

Summary Thirteen nuclear asymmetric hybrids were regenerated under selective conditions following fusion of chlorophyll-deficient protoplasts from cultivated tomato (Lycopersicon esculentum Mill.) and -(-irradiated protoplasts from the wild species Lycopersicon peruvianum var. dentatum Dun. All hybrid plants were classified as being asymmetric based on morphological traits, chromosome numbers and isozyme patterns. The majority of the hybrids inherited Lycopersicon peruvianum var. dentatum chloroplasts. Mitochondrial DNA analysis revealed mixed mitochondria populations deriving from both parents in some of the hybrids and rearranged mitochondrial DNA in others. The asymmetric hybrids express some morphological traits that are not found in either of the parental species. Fertile F₁ plants were obtained after self-pollination of the asymmetric hybrids in four cases. The results obtained confirm the potential of asymmetric hybridization as a new source of genetic variation, and as a method for transferring of a part of genetic material from donor to recipient, and demonstrate that it is possible to produce fertile somatic hybrids by this technique.     

Drivers of microbially and plant-derived carbon in topsoil and subsoil

Plant- and microbially derived carbon (C) are the two major sources of soil organic matter (SOM), and their ratio impacts SOM composition, accumulation, stability, and turnover. The contributions of and the key factors defining the plant and microbial C in SOM along the soil profile are not well known. By leveraging nuclear magnetic resonance spectroscopy and biomarker analysis, we analyzed the plant and microbial C in three soil types using regional-scale sampling and combined these results with a meta-analysis. Topsoil (0–40 cm) was rich in carbohydrates and lignin (38%–50%), whereas subsoil (40–100 cm) contained more proteins and lipids (26%–60%). The proportion of plant C increases, while microbial C decreases with SOM content. The decrease rate of the ratio of the microbially derived C to plant-derived C (C_M:P) with SOM content was 23%–30% faster in the topsoil than in the subsoil in the regional study and meta-analysis. The topsoil had high potential to stabilize plant-derived C through intensive microbial transformations and microbial necromass formation. Plant C input and mean annual soil temperature were the main factors defining C_M:P in topsoil, whereas the fungi-to-bacteria ratio and clay content were the main factors influencing subsoil C_M:P. Combining a regional study and meta-analysis, we highlighted the contribution of plant litter to microbial necromass to organic matter up to 1-m soil depth and elucidated the main factors regulating their long-term preservation.     

Response of root respiration and root exudation to alterations in root C supply and demand in wheat

Rising atmospheric CO₂ concentrations have highlighted the importance of being able to understand and predict C fluxes in plant-soil systems. We investigated the responses of the two fluxes contributing to below-ground efflux of plant root-dependent CO₂, root respiration and rhizomicrobial respiration of root exudates. Wheat (Triticum aestivum L., var. Consort) plants were grown in hydroponics at 20°C, pulse-labelled with ¹⁴CO₂ and subjected to two regimes of temperature and light (12 h photoperiod or darkness at either 15°C or 25°C), to alter plant C supply and demand. Root respiration was increased by temperature with a Q ₁₀ of 1.6. Root exudation was, in itself, unaltered by temperature, however, it was reduced when C supply to the roots was reduced and demand for C for respiration was increased by elevated temperature. The rate of exudation responded much more rapidly to the restriction of C input than did respiration and was approximately four times more sensitive to the decline in C supply than respiration. Although temporal responses of exudation and respiration were treatment dependent, at the end of the experimental period (2 days) the relative proportion of C lost by the two processes was conserved despite differences in the magnitude of total root C loss. Approximately 77% of total C and 67% of ¹⁴C lost from roots was accounted for by root respiration. The ratio of exudate specific activity to CO₂ specific activity converged to a common value for all treatments of 2, suggesting that exudates and respired CO₂were not composed of C of the same age. The results suggest that the contributions of root and rhizomicrobial respiration to root-dependent below-ground respiration are conserved and highlight the dangers in estimating short-term respiration and exudation only from measurements of labelled C. The differences in responses over time and in the age of C lost may ultimately prove useful in improving estimates of root and rhizomicrobial respiration.     

Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.