Birgit Andersen

Responses of barley root and shoot proteomes to long-term nitrogen deficiency, short-term nitrogen starvation and ammonium.

Cereals are major crops worldwide, and improvement of their nitrogen use efficiency is a crucial challenge. In this study proteins responding to N supply in barley roots and shoots were analysed using a proteomics approach, to provide insight into mechanisms of N uptake and assimilation. Control plants grown hydroponically for 33 d with 5 mm nitrate, plants grown under N deficiency (0.5 mm nitrate, 33 d) or short-term N starvation (28 d with 5 mm nitrate followed by 5 d with no N source) were compared. N deficiency caused changes in C and N metabolism and ascorbate-glutathione cycle enzymes in shoots and roots. N starvation altered proteins of amino acid metabolism in roots. Both treatments caused proteome changes in roots that could affect growth. Shoots of plants grown with ammonium as N source (28 d with 5 mm nitrate followed by 5 d with 5 mm ammonium) showed responses similar to N deficient shoots, characterized by turnover of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and increases in proteins of the chloroplastic transcription and translation machinery. Identified proteins in 67 and 49 varying spots in roots and shoots respectively, corresponded to 62 functions and over 80 gene products, considerably advancing knowledge of N responses in barley.

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination‐related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.

Nonspecific facilitation of responses to transcranial magnetic stimulation

We examined the effect of facial muscle contraction and eye movements on motor evoked potentials (MEPs) from the abductor pollicis brevis muscle (APB) evoked by transcranial magnetic stimulation (TMS). The hypothesis was that activity of large cortical regions (face) influences the excitability of spinal motoneurons via cortical or subcortical pathways. MEPs were recorded in 12 healthy subjects during the following conditions: (1) rest; (2) facial muscle contraction; (3) eye movements; (4) 10% precontraction of the target muscle; and (5) simultaneous target muscle precontraction and facial muscle contraction. In 9 subjects, spinal motoneuron excitability was assessed by measurements of F waves during the same facilitation maneuvers. Activation of eye and facial muscles clearly facilitated MEPs from the APB. The facilitation of MEP size during nonspecific maneuvers was almost similar to that obtained by target muscle precontraction, whereas shortening of latencies was significantly smaller. The occurrence and amplitude of F waves increased in parallel with MEP size during specific and nonspecific facilitation, pointing to spinal motoneuronal threshold changes as a potential facilitatory mechanism by facial and eye muscle activation. The different MEP latencies during specific and nonspecific facilitation were not explained by different spinal motoneuron excitability, but raise the possibility that supraspinal mechanisms contributed to nonspecific facilitation.

Monthly oral methylprednisolone pulse treatment in progressive multiple sclerosis

Background: There is a large unmet need for treatments for patients with progressive multiple sclerosis (MS). Phase 2 studies with cerebrospinal fluid (CSF) biomarker outcomes may be well suited for the initial evaluation of efficacious treatments. Objective: To evaluate the effect of monthly oral methylprednisolone pulse treatment on intrathecal inflammation in progressive MS. Methods: In this open-label phase 2A study, 15 primary progressive and 15 secondary progressive MS patients received oral methylprednisolone pulse treatment for 60 weeks. Primary outcome was changes in CSF concentrations of osteopontin. Secondary outcomes were other CSF biomarkers of inflammation, axonal damage and demyelination; clinical scores; magnetic resonance imaging measures of disease activity, magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI); motor evoked potentials; and bone density scans. Results: We found no change in the CSF concentration of osteopontin, but we observed significant improvement in clinical scores, MTR, DTI and some secondary CSF outcome measures. Adverse events were well-known side effects to methylprednisolone. Conclusion: Monthly methylprednisolone pulse treatment was safe, but had no effect on the primary outcome. However, improvements in secondary clinical and MRI outcome measures suggest that this treatment regimen may have a beneficial effect in progressive MS.

Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC-MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation and included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment.

Crystallization and preliminary X‐ray analysis of β‐­alanine synthase from the yeast Saccharomyces kluyveri

In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. BIOLOGICAL SIGNIFICANCE The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications.