Characterisation of the tryptophan synthase alpha subunit in maize

Background  

In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB.     

Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an <Emphasis Type="Italic">in vitro</Emphasis>study

Background  

Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.     

Geographical and technological differences in life cycle inventories shown by the use of process models for waste incinerators part I. technological and geographical differences

Goal and Background  Geographical and technological differences in Life Cycle Inventory data are an important source for uncertainty in the result of Life Cycle Assessments. Knowledge on their impact on the result of an LCA is scarce, and also knowledge on how to manage them in an LCA case study. Objective  Goal of this paper is to explore these differences for municipal solid waste incinerator plants, and to develop recommendations for managing technological and geographical differences. Methodology  The paper provides a definition of technological and geographical differences, and analyses their possible impacts. In a case study, the differences are caused intentionally in ‘games’, by virtually transplanting incineration plants to a different location and by changing parameters such as the composition of the waste input incinerated. The games are performed by using a modular model for municipal solid waste incinerator plants. In each case, an LCA including an Impact Assessment is calculated to trace the impact of these changes, and the results are compared. Conclusions  The conclusions of the paper are two-fold: (1) reduce the differences in inventory data where their impact on the result is high; where it is possible reducing them to a great extent, and the effort for performing the change acceptable; in the case of incineration plants: Adapt the flue gas treatment, especially a possible DeNOx step, to the real conditions; (2) make use of modular process models that allow adapting plant parameters to better meet real conditions, but be aware of possible modelling errors. The paper invites the scientific community to validate the model used for a waste incinerator plant, and suggest putting up similar models for other processes, preferably those of similar relevance for Life Cycle Inventories.     

PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA

We investigated the PvuII endonuclease with its cognate DNA by means of molecular dynamics simulations. Comparing the complexed DNA with a reference simulation of free DNA, we saw structural changes at the scissile phosphodiester bond. At this GpC step, the enzyme induces the highest twist and axial rise, inclination is increased and the minor groove widened. The distance between the scissile phosphate group and the phosphate group of the following thymine base is shortened significantly, indicating a substrate-assisted catalysis. A feasible reason for this vicinity is the catalytically important amino acid residue lysine 70, which bridges the free oxygen atoms of the successive phosphate groups. Due to this geometry, a compact reaction pocket is formed where a water molecule can be held, thus bringing the reaction partners for hydrolysis into contact. The O1-P-O2 angle of the scissile nucleotide is decreased, probably due to a complexation of the negative oxygen atoms through protein and solvent contacts.     

Identification of oxidized methionine residues in peptides containing two methionine residues by derivatization and matrix-assisted laser desorption/ionization mass spectrometry

Oxidation of methionine residues in peptides and proteins occurs in vivo or may be an artifact resulting from purification steps. We present a three step method for the localization of methionine sulfoxides in peptides with two methionine residues. In the first step, the N-terminus as well as other reactive side chain functions are blocked by acetylation. The resulting protected peptides are cleaved by cyanogen bromide. The cleavage does not occur at methionine sulfoxide but only at reduced methionine residues forming new amino termini. The newly formed amino group is then derivatized with a bromine containing compound in the last step of the procedure. The resulting peptide can easily be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using both the characteristic isotope pattern of the halogen and the metastable loss of methanesulfenic acid from oxidized residues. This procedure allows the unequivocal localization of oxidized methionines even in complex peptide mixtures.     

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.     

Availability of optimal‐sized prey affects global distribution patterns of the golden eagle Aquila chrysaetos

Climate and landscape change are expected to significantly affect trophic interactions, which will especially harm top predators such as the golden eagle Aquila chrysaetos. Availability of optimal prey is recognized to influence reproductive success of raptors on a regional scale. For the golden eagle, medium‐sized prey species between 0.5 and 5 kg are widely considered to be optimal prey during the breeding season, whereas smaller and larger species are deemed as energetically sub‐optimal. However, knowledge about the effects of optimal prey availability is still scarce on larger scales. To decrease this apparent knowledge gap, we combined biogeographical information on range margins with information about the foraging behaviour and reproductive success of golden eagles from 67 studies spanning the Northern Hemisphere. We hypothesized that availability of optimal prey will affect foraging behaviour and breeding success and, thus, distribution patterns of the golden eagle not only on a local but also on a continental scale. We correlated the diet breadth quantifying foraging generalism, breeding success and proportions of small (< 0.5 kg), medium (0.5–5 kg) and large‐sized (> 5 kg) prey species within the diet with the minimum distance of the examined eagles to the actual species distribution boundary. Closer to the range edge, we observed decreased proportions of medium‐sized prey species and decreasing breeding success of golden eagles. Diet breadth as well as proportions of small and large‐sized prey species increased, however, towards the range edge. Thus, availability of optimal‐sized prey species seems to be a crucial driver of foraging behaviour, breeding success and distribution of golden eagles on a continental scale. However, underlying effects of landscape characteristics and human influence on optimal prey availability has to be investigated in further large‐scale studies to fully understand the major threats facing the golden eagle and possibly other large terrestrial birds of prey.     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Development of a Monoclonal Antibody Detection Assay for Species-Specific Identification of Abalone

Species identification based on biochemical and molecular techniques has a broad range of applications. These include compliance enforcement, the management and conservation of marine organisms, and commercial quality control. Abalone poaching worldwide and illegal trade in abalone products have increased mainly because of the attractive prices obtained and caused a sharp decline in stocks. Alleged poachers have been acquitted because of lack of evidence to correctly identify species. Therefore, a robust method is required that would identify tissue of abalone origin to species level. The aim of this study was to develop immunologic techniques, using monoclonal and polyclonal antibodies, to identify 10 different abalone species and subspecies from South Africa, the United States, Australia, and Japan. The combination of 3 developed monoclonal antibodies to South African abalone (Haliotis midae) enabled differentiation between most of the 10 species including the subspecies H. diversicolor supertexta and H. diversicolor diversicolor. In a novel approach, using antibodies of patients with allergy to abalone, the differentiation of additional subspecies, H. discus discus and H. discus hannai, was possible. A field-based immunoassay was developed to identify confiscated tissue of abalone origin.