Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.     

Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers. Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.     

Functional composition of plant communities determines the spatial and temporal stability of soil microbial properties in a long‐term plant diversity experiment

Stable provisioning of ecosystem functions and services is crucial for human well‐being in a changing world. Two essential ecological components driving vital ecosystem functions in terrestrial ecosystems are plant diversity and soil microorganisms. In this study, we tracked soil microbial basal respiration and biomass over a time period of 12 years in a grassland biodiversity experiment (the Jena Experiment) and examined the role of plant diversity and plant functional group composition for the spatial and temporal stability of soil microbial properties (basal respiration and biomass) in bulk‐soil. Spatial and temporal stability were calculated as the inverse coefficient of variation (CV^?1) of soil microbial respiration and biomass measured from soil samples taken over space and time, respectively. We found that 1) plant species richness consistently increased soil microbial properties after a time lag of four years since the establishment of the experimental plots, 2) plant species richness had minor effects on the spatial stability of soil microbial properties, whereas 3) the functional composition of plant communities significantly affected spatial stability of soil microbial properties, with legumes and tall herbs reducing both the spatial stability of microbial respiration and biomass, while grasses increased the latter, and 4) the effect of plant diversity on temporal stability of soil microbial properties turned from being negative to neutral, suggesting that the recovery of soil microbial communities from former arable land‐use takes more than a decade. Our results highlight the importance of plant functional group composition for the spatial and temporal stability of soil microbial properties, and hence for microbially‐driven ecosystem processes, such as decomposition and element cycling, in temperate semi‐natural grassland.     

Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,¹⁵N values, and ¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,¹⁵N_nitrate and¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,¹⁵N_nitrate values of lessthan +5, and ¹⁸O_nitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with ¹⁵N_nitratevalues between +5 and +8 and¹⁸O_nitrate values generallybelow +15. Correlations between nitrateconcentrations, ¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated ¹⁵N_nitrate and¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Plant diversity does not buffer drought effects on early‐stage litter mass loss rates and microbial properties

Human activities are decreasing biodiversity and changing the climate worldwide. Both global change drivers have been shown to affect ecosystem functioning, but they may also act in concert in a non‐additive way. We studied early‐stage litter mass loss rates and soil microbial properties (basal respiration and microbial biomass) during the summer season in response to plant species richness and summer drought in a large grassland biodiversity experiment, the Jena Experiment, Germany. In line with our expectations, decreasing plant diversity and summer drought decreased litter mass loss rates and soil microbial properties. In contrast to our hypotheses, however, this was only true for mass loss of standard litter (wheat straw) used in all plots, and not for plant community‐specific litter mass loss. We found no interactive effects between global change drivers, that is, drought reduced litter mass loss rates and soil microbial properties irrespective of plant diversity. High mass loss rates of plant community‐specific litter and low responsiveness to drought relative to the standard litter indicate that soil microbial communities were adapted to decomposing community‐specific plant litter material including lower susceptibility to dry conditions during summer months. Moreover, higher microbial enzymatic diversity at high plant diversity may have caused elevated mass loss of standard litter. Our results indicate that plant diversity loss and summer drought independently impede soil processes. However, soil decomposer communities may be highly adapted to decomposing plant community‐specific litter material, even in situations of environmental stress. Results of standard litter mass loss moreover suggest that decomposer communities under diverse plant communities are able to cope with a greater variety of plant inputs possibly making them less responsive to biotic changes.     

Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.In the last few years, autotrophic nitrogen removal via partial nitritation and anoxic ammonium oxidation (anammox) has evolved from lab- to full-scale treatment of nitrogenous wastewaters with a low biodegradable organic compound content, and this evolution has been driven mainly by a significant decrease in the operational costs compared to the costs of conventional nitrification and heterotrophic denitrification (11, 23). Oxygen-limited autotrophic nitrification and denitrification (OLAND) is one of the autotrophic processes used and is a one-stage procedure; i.e., partial nitritation and anammox occur in the same reactor (30). The “functional” autotrophic microorganisms in OLAND include aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB). With oxygen, AerAOB oxidize ammonium to nitrite (nitritation), and with the nitrite AnAOB oxidize the residual ammonium to form dinitrogen gas and some nitrate (anammox). Additional aerobic nitrite oxidation to nitrate (nitratation) by nitrite-oxidizing bacteria (NOB) lowers the nitrogen removal efficiency, but it can, for instance, be prevented at low dissolved oxygen (DO) levels because the oxygen affinity of AerAOB is higher than that of NOB (16). Reactor configurations for the OLAND process can be based on suspended biomass growing in aggregates, like that in a sequencing batch reactor (SBR) (37) or a gas lift or upflow reactor (32). For suspended-growth systems there are two important challenges: biomass retention and equilibrated microbial activities.High biomass retention efficiency is a prerequisite in anammox technologies because of the slow growth of AnAOB (33). In suspended biomass systems, settling properties determine the retention of biomass and are related to the microbial aggregate morphology (floc or granule) and size. Granules can be defined as compact and dense aggregates with an approximately spherical external appearance that do not coagulate under decreased hydrodynamic shear conditions and settle significantly faster than flocs (18). Toh and coworkers calculated a lower sludge volume index for aerobic granules than for aerobic flocs and also showed that there was an increase in the settling velocity with increasing granule size (35). Hence, in terms of physical properties, large granules are preferable for suspended-growth applications.OLAND aggregate size not only influences settling properties but also affects the proportion of microbial nitrite production and consumption; lower AerAOB activity and higher AnAOB activity were observed with larger aggregates (25, 37). Theoretically, a microbial aggregate with equal nitrite production and nitrite consumption can remove ammonium autonomously, because of its independence from other aggregates for acquisition and conversion of nitrite. Hence, with an increasing aggregate size and thus with a decreasing ratio of nitrite production to nitrite consumption, three functional categories of aggregates can be distinguished: nitrite sources, autonomous nitrogen removers, and nitrite sinks. Because minimal nitrite accumulation is one of the prerequisites for high nitrogen removal efficiency in OLAND reactors, the presence of excess small aggregates is undesirable (9, 37).Although large granular aggregates are desirable for biomass retention and activity balance, so far no formation mechanisms have been proposed for OLAND granules, in contrast to the well-studied anaerobic (13) and aerobic (1) granules. In order to determine general and environment-specific determinants for aggregate size and architecture, three suspended-growth OLAND reactors with different inoculation and operation (mixing and aeration) parameters were selected, and these reactors were designated reactors A, B, and C (Table ​(Table1).1). The first objective of this study was to gain more insight into the relationship between OLAND aggregate size, AerAOB and AnAOB abundance, and the activity balance. The second objective was to propose pathways for aggregation and granulation by relating (dis)similarities in aggregate size distribution, morphology, and architecture to differences in reactor inoculation and operation.

TABLE 1.

Overview of the three OLAND reactor systems from which suspended biomass samples were obtained

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.