Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

The effect of fasting on the activation in vivo of the insulin receptor kinase.

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.     

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis

WW domain binding protein 1‐like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6‐RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non‐haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4‐family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l‐deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.     

Vitamin D receptor BsmI polymorphism modulates soy intake and 25-hydroxyvitamin D supplementation benefits in cardiovascular disease risk factors profile

Vitamin D receptor polymorphisms may predispose that not all individuals could have benefits from the nutritional supplementation of 25-hydroxyvitamin D. Furthermore, vitamin D-related cardiovascular effects may also be influenced by soy isoflavones considered endocrine regulators of cardiovascular homeostasis. To find possible gene–diet interactions by evaluating individualized lipid metabolism benefits from an increase in soy and 25-hydroxyvitamin D intake, 106 healthy individuals, genotyped for vitamin D receptor (VDR) gene polymorphism rs1544410 (BsmI) were randomly assigned to either no intake, to daily 250?mL or 500?mL of a 25-hydroxyvitamin D supplemented SB for 2 months. The soybean beverage induced differences in cardiovascular risk factors (lipid profile, blood pressure, TNFα and MCP-1), as well as vitamin D metabolites in a dose-gene-dependent relation. Thus, VDR BsmI polymorphism affected individual response being the GG genotype the ones that showed dose-dependent manner responsiveness in the reduction in total cholesterol, LDL and triglycerides in comparison with the AA/AG genotype. These differences were associated with increased plasma levels of 1α,25-dyhydroxyvitamin D3 in the carriers of the GG genotype. It was concluded that metabolic response to 25-hydroxyvitamin D and soybean supplementation is dependent on VDR BsmI GG genotype due to a higher conversion rate from vitamin D precursors.     

Mixtures with grass litter may hasten shrub litter decomposition after shrub encroachment into mountain grasslands

Aims

Shrub encroachment in mesic grasslands alters the identity and quality of litters entering the system. As litter from shrubs and grasses can differ in their quality, this can lead to differences in litter decomposition by the direct effect of quality, but also to litter interaction during decomposition. The objective of this study was to examine the occurrence of non-additive effects of litter mixtures on the decomposition rates of legume shrub litter (poor in P) or conifer shrub litter (poor in N) and grass litter.

Methods

In addition to single litter type litterbags for the three species, we mixed litters of each pair of possible combinations to determine the influence of each species on mass loss. Litterbags were placed in the field and collected after 1, 6, 8, 12 and 24 months. In each collection, litter of each species remaining in mixed bags was separated, dry weighed and analyzed for C, N and P.

Results

With respect to shrub litter decomposing alone, mass loss of shrub litter when mixed with grass showed a 9–10 % increase in decomposition rate for conifer and a 3 % increase for legume litter. These litter mixture effects varied with time and they were detected after a decomposition period of 1 year in legume litter and of 2 years in conifer litter.

Conclusions

Grass litter hastened conifer and legume litter decomposition in leaf litter mixtures, at least during the first stages of the process. The potential consequences of this result to alter litter accumulation patterns and thus carbon sequestration rates after shrub encroachment into grasslands will depend on whether the observed trends are maintained in the advanced decomposition stages.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Individual closed chamber: an alternative method for quantifying 14C in both labeled organic and inorganic carbon substrates

Incubation of ¹⁴C-labeled substrates continues to be a widely used procedure in soil organic matter (OM) research due to its sensitiveness. When the labeling is found in liquid fractions (soil extracts, hydrolysates), ¹⁴C can be easily quantified by using an aliquot for scintillation counting. For this reason, converting a solid carbon sample into liquid form is a typical step for accurate ¹⁴C analysis. We have developed an alternative method to carry out this step, which uses standard glass hardware and does not require complex laboratory facilities. Carbon (both in organic or inorganic forms) is converted into CO₂ within a reaction vessel connected to a Twisselmann’s extractor with an alkali trap inside. This forms an individual closed chamber (ICC) for each sample, thus eliminating the risk of cross-contaminations. The alkali solution adsorbs the evolved CO₂ within the closed system, and the excess of pressure is easily overcome by the use of a balloon. We tested the procedure on a set of substrates and two contrasting soils, checking also the effect of different sample loads (from 20 to 160 mg C) on the CO₂ recovery of the process. The percentage of carbon recovered into the alkali (i.e. the efficiency of the process) ranged from 92% for the inorganic C to 93–95% for the organic C method, the latter being sensitive to the amount of sample used for analysis. The ICC method can be successfully applied to analyze ¹⁴C-labeling in both carbonates and OM from solid samples, thus representing an alternative method to some established protocols, and it is suitable for substrates with low or very low ¹⁴C contents, in which high volumes of sample must be analyzed in order to guarantee representative results.