Osmotische Messungen an hochkonzentrierten Lösungen von Rinderserumalbumin

Zusammenfassung In der vorliegenden Arbeit werden osmotische Messungen an Rinderserumalbuminlösungen verschiedener Konzentrationen beschrieben. Die Messungen erstreckten sich auf Konzentrationen bis zu etwa 25%, wie sie in lebenden Zellen vorkommen. Aus den Meßergebnissen wurde die Änderung des chemischen Potentials berechnet. Es wurde versucht, diese Änderung mit Hilfe der Theorie der athermischen Lösungen zu erklären. In dem vorliegenden Fall gelten innerhalb des Konzentrationsbereiches, für den die Rechnung durchgeführt werden konnte, die Voraussetzungen der athermischen Lösung. Das Molekulargewicht des verwendeten Rinderserumalbumins ergab sich auf Grund der osmotischen Messungen zu 73000 ± 10%.     

Microanalysis of Plant Cell Wall Polysaccharides

Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.     

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation² and other products such as biocomposite materials⁶. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin⁵ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation⁵. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose⁷. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I³.     

Focus Issue on Plant Cell Walls: Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.Purple acid phosphatase (PAP) belongs to a large family of dinuclear metalloenzymes (LeBansky et al., 1992; Klabunde et al., 1996) and catalyzes the hydrolysis of a wide range of phosphate esters. It is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron (III) charge transfer transition (Antanaitis et al., 1983). Arabidopsis (Arabidopsis thaliana) contains a large family of PAPs composed of 29 genes, 28 of which have signal peptides that potentially transfer to the wall and/or vacuole. Only a few functions have been suggested for these phosphatases: AtPAP15 seems to modulate ascorbic acid biosynthesis (Zhang et al., 2008), and AtPAP17 may play a role in the metabolism of reactive oxygen species (del Pozo et al., 1999). In other plant species, soybean (Glycine max) GmPAP3 is induced by NaCl stress but not by phosphorus deficiency (Liao et al., 2003), tomato (Solanum lycopersicum) PAP may release phosphate from extracellular phosphate ester under phosphate starvation (Bozzo et al., 2002), and tobacco (Nicotiana tabacum) NtPAP12 could be involved in the deposition of β-glucan (Kaida et al., 2003, 2009; Sano et al., 2003). Mammalian PAPs, which are secretory enzymes, may be involved in iron transport (Nuttleman and Roberts, 1990), generation of reactive oxygen species (Sibille et al., 1987), and bone resorption (Ek-Rylander et al., 1994).We previously demonstrated that the activities of cellulose and callose synthases are enhanced by overexpression of NtPAP12 in tobacco cells (Kaida et al., 2009). The phosphorylation/dephosphorylation process in those synthases may occur directly on the catalytic subunit itself, which has been predicted to be located on the cytoplasmic side of the plasma membrane (Nühse et al., 2004; Taylor, 2007). This is not compatible with the cell wall localization of NtPAP12. The data also indicate that phosphorylation may play a role in regulating the turnover of cellulose synthase by proteolysis through a proteasome-dependent pathway (Taylor, 2007), which again implies a cytoplasmic phosphorylation event. Thus, we suggested that NtPAP12 could be involved in the regulation of cellulose synthase activity, either by acting on an unidentified membrane protein or by enhancing its activity with an effector, which can lead to the promotion of cellulose synthesis. Nevertheless, this phosphatase may be involved in the activation of synthases indirectly by acting on either apoplastic proteins or unidentified membrane proteins, since the level of activation for glucan synthases was only a 2- to 3-fold increase in the transgenic tobacco cells overexpressing NtPAP12 compared with wild-type cells.The extracellular phosphorylation network has been proposed by proteomic analysis of Arabidopsis cells due to the identification of phosphorylated Tyr residues in xyloglucanase, putative lectin receptor-like kinase, and putative chitinase (Ndimba et al., 2003). The change in phosphorylation status was also identified in the extracellular peroxidase in maize (Zea mays) cells (Chivasa et al., 2005b). Another analysis has indicated that some potential phosphorylated proteins might be present in the apoplastic space during wall regeneration (Kwon et al., 2005). We previously showed that tobacco PAP had a higher catalytic efficiency for Tyr phosphopeptides (k_cat/K_m = 1,093–1,335) than for ATP (k_cat/K_m = 333) and p-nitrophenyl-phosphate (k_cat/K_m = 379), suggesting that the enzyme could dephosphorylate the phosphoryl residues of proteins in vivo (Kaida et al., 2008). There is still much to be learned, however, including the role that phosphorylation plays in the functions of these proteins. It is possible, for example, that extracellular PAPs might modify the functions of the phosphoproteins by dephosphorylating those proteins in the apoplasts, but to date no evidence has been reported demonstrating this activity. In this study, we searched for substrates of PAP using phosphoproteomic analyses of apoplastic proteins in tobacco cells.     

The growth of jellyfishes

To date, a disparate array of concepts and methods have been used to study the growth of jellyfish, with the result that few generalities have emerged which could help, e.g., in predicting growth patterns in unstudied species. It is shown that this situation can be overcome by length-frequency analysis (LFA), applied to jellyfish bell diameter (i.e., “length”) frequency data. A selection of LFA methods (ELEFAN, Wetherall plots and length-converted catch curves, all implemented in the FiSAT software) is applied here to 34 sets of bell diameter frequency data of jellyfish. This led to the estimates of parameters of the von Bertalanffy growth function (VBGF), which, especially in its seasonal form, was found to fit the available size-frequency data reasonably well. We also obtained numerous estimates of mortality, useful for modeling the life history of jellyfish. Finally, by scaling their asymptotic weight (W _∞, a parameter of the VBGF) to the weight they would have if they had the same water content as fish, we show that most jellyfish grow at the same rate as small fishes (guppies and anchovies). As in fish, the VBGF parameters K and W _∞, when plotted in a double logarithmic (“auximetric”) plot, tend to cluster into ellipsoid shapes, which increase in area when shifting from species to genera, families, etc. If validated by subsequent studies, auximetric plots for jellyfish would provide a powerful tool for testing comparative hypotheses on jellyfish life history. Guest editors: K. A. Pitt & J. E. Purcell Jellyfish Blooms: Causes, Consequences, and Recent Advances     

The spatial expansion and ecological footprint of fisheries (1950 to present)

Using estimates of the primary production required (PPR) to support fisheries catches (a measure of the footprint of fishing), we analyzed the geographical expansion of the global marine fisheries from 1950 to 2005. We used multiple threshold levels of PPR as percentage of local primary production to define 'fisheries exploitation' and applied them to the global dataset of spatially-explicit marine fisheries catches. This approach enabled us to assign exploitation status across a 0.5° latitude/longitude ocean grid system and trace the change in their status over the 56-year time period. This result highlights the global scale expansion in marine fisheries, from the coastal waters off North Atlantic and West Pacific to the waters in the Southern Hemisphere and into the high seas. The southward expansion of fisheries occurred at a rate of almost one degree latitude per year, with the greatest period of expansion occurring in the 1980s and early 1990s. By the mid 1990s, a third of the world's ocean, and two-thirds of continental shelves, were exploited at a level where PPR of fisheries exceed 10% of PP, leaving only unproductive waters of high seas, and relatively inaccessible waters in the Arctic and Antarctic as the last remaining 'frontiers.' The growth in marine fisheries catches for more than half a century was only made possible through exploitation of new fishing grounds. Their rapidly diminishing number indicates a global limit to growth and highlights the urgent need for a transition to sustainable fishing through reduction of PPR.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula

The model legume Medicago truncatula possesses a single outward Shaker K⁺ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K⁺ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K⁺ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch‐clamp experiments on root hair protoplasts, besides the Shaker‐type slowly activating outwardly rectifying K⁺ conductance encoded by MtGORK, a second K⁺‐permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock‐out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K⁺ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod‐factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.