Chlorierte Kohlenwasserstoffe in Brutv?geln aus dem Binnenland Niedersachsens

Zusammenfassung Rückstände chlorierter Kohlenwasserstoffe in Eiern und Lebern von im Binnenland Niedersachsens brütenden Vogelarten — Feldsperling, Mehlschwalbe, Weißstorch, Graureiher, Saatkrähe, Stockente und andere Arten — werden angegeben und deren Abhängigkeit von Brutort, Nahrung und Zugverhalten diskutiert.

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Chlorinated hydrocarbons of some bird species breeding in the inland of Lower Saxony (FRG)

Summary Residues of chlorinated hydrocarbons in eggs and livers of some bird species — Tree Sparrow, House Martin, White Stork, Heron, Rook, Mallard, and further species — are presented. The dependence on place of breeding, food web, and migration is discussed.

     

Two recurrent nonsense mutations and a 4 bp deletion in a quasi-symmetric element in exon 37 of the NF1 gene

We screened a total of 92 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 37 of the NF1 gene, by using temperature gradient gel electrophoresis. Two novel mutations were found: a 4 bp deletion in a so-called quasi-symmetric element (6789delTTAC) and a recurrent nonsense mutation, which was identified in two unrelated patients, at codon 2264 (C6792A). The independent origin of the latter mutation in two families was confirmed by haplotype analysis. The nonsense mutation and the 4 bp deletion are both predicted to lead to a truncated protein product lacking the Cterminal 20% (aproximately) of its sequence. The occurrence of three independent mutations among 92 NF1 patients at codons 2263–2264 (exon 37) suggests that a specific search for mutations in this area should be undertaken in screening programs for NF1 mutations.     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).     

Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor

The Kv-like (potassium voltage-dependent) K⁺ channels at the plasma membrane, including the inward-rectifying KAT1 K⁺ channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K⁺ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K⁺ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.Plant cells utilize the potassium ion (K⁺) to maintain hydrostatic (turgor) pressure, to drive irreversible cell expansion for growth, and to facilitate reversible changes in cell volume during stomatal movements. Potassium uptake and its circulation throughout the plant relies both on high-affinity, H⁺-coupled K⁺ transport (Quintero and Blatt, 1997; Rubio et al., 2008) and on K⁺ channels to facilitate K⁺ ion transfer across cell membranes. Uptake via K⁺ channels is thought to be responsible for roughly 50% of the total K⁺ content of the plant under most field conditions (Spalding et al., 1999; Rubio et al., 2008; Amtmann and Blatt, 2009). K⁺ channels confer on the membranes of virtually every tissue distinct K⁺ conductances and regulatory characteristics (Véry and Sentenac, 2003; Dreyer and Blatt, 2009). Their characteristics are thus of interest for engineering directed to manipulating K⁺ flux in many aspects of plant growth and cellular homeostasis. The control of K⁺ channel gating has been identified as the most promising target for the genetic engineering of stomatal responsiveness (Lawson and Blatt, 2014; Wang et al., 2014a), based on the recent development of quantitative systems models of guard cell transport and metabolism (Chen et al., 2012b; Hills et al., 2012; Wang et al., 2012). By contrast, modifying the expression and, most likely, the population of native K⁺ channels at the membrane was found to have no substantial effect on stomatal physiology (Wang et al., 2014b).The Kv-like K⁺ channels of the plant plasma membrane (Pilot et al., 2003; Dreyer and Blatt, 2009) share a number of structural features with the Kv superfamily of K⁺ channels characterized in animals and Drosophila melanogaster (Papazian et al., 1987; Pongs et al., 1988). The functional channels assemble from four homologous subunits and surround a central transmembrane pore that forms the permeation pathway (Daram et al., 1997). Each subunit comprises six transmembrane α-helices, designated S1 to S6, and both N and C termini are situated on the cytosolic side of the membrane (Uozumi et al., 1998). The pore or P loop between the S5 and S6 α-helices incorporates a short α-helical stretch and the highly conserved amino acid sequence TxGYGD, which forms a selectivity filter for K⁺ (Uozumi et al., 1995; Becker et al., 1996; Nakamura et al., 1997). The carbonyl oxygen atoms of these residues in all four K⁺ channel subunits face inward to form coordination sites for K⁺ ions between them (Doyle et al., 1998; Jiang et al., 2003; Kuo et al., 2003; Long et al., 2005) and a multiple-ion pore (Thiel and Blatt, 1991) such that K⁺ ions pass through the selectivity filter as if in free solution. The plant channels are also sensitive to a class of neurotoxins that exhibit high specificity in binding around the mouth of the channel pore (Obermeyer et al., 1994).These K⁺ channels also share a common gating mechanism. Within each subunit, the first four α-helices form a quasiindependent unit, the voltage sensor domain (VSD), with the S4 α-helix incorporating positively charged (Arg or Lys) residues regularly positioned across the lipid bilayer and transmembrane electric field. Voltage displaces the S4 α-helix within the membrane and couples rotation of the S5 and S6 α-helices lining the pore, thereby opening or closing the channel (Sigworth, 2003; Dreyer and Blatt, 2009). For outward-rectifying channels, such as the mammalian Kv1.2 and the D. melanogaster Shaker K⁺ channels, an inside-positive electric field drives the positively charged, S4 α-helix outward (the up position), which draws on the S4-S5 linker to open the pore. This simple expedient of a lever and string secures current flow in one direction by favoring opening at positive, but not negative, voltages. This same model applies to the Arabidopsis (Arabidopsis thaliana) Kv-like K⁺ channels, including outward rectifiers that exhibit sensitivity to external K⁺ concentration (Blatt, 1988; Blatt and Gradmann, 1997; Johansson et al., 2006), and it serves equally in the gating of inward-rectifying K⁺ channels such as KAT1, which gates open at negative voltages (Dreyer and Blatt, 2009).Studies of KAT1 gating (Latorre et al., 2003; Lai et al., 2005) have indicated that the S4 α-helix of the channel most likely undergoes very similar conformational changes with voltage as those of the mammalian and Shaker K⁺ channels. These findings conform with the present understanding of the evolution of VSD structure (Palovcak et al., 2014) and the view of a common functional dynamic to its molecular design. It is likely, therefore, that a similar electrostatic network occurs in KAT1 to stabilize the VSD. Crucially, however, experimental evidence in support of such a network has yet to surface. Electrostatic countercharges and the hydration of amino acid side chains between the α-helices within the VSDs of mammalian and Shaker K⁺ channel models are important for the latch-like stabilization of the so-called down and up states of these channels (Tao et al., 2010; Pless et al., 2011). Nonetheless, some studies (Gajdanowicz et al., 2009; Riedelsberger et al., 2010) have pointed to subtle differences in the structure of KAT1 that relate to the VSD.We have explored the electrostatic network of the KAT1 VSD through site-directed mutagenesis to manipulate the voltage dependence of KAT1, combining these studies with molecular dynamic simulations previously shown to accommodate the plant VSDs and their hydration during gating transitions (Gajdanowicz et al., 2009; Garcia-Mata et al., 2010). We report here that gating of KAT1 is sensitive to manipulations affecting a set of electrostatic charge transfer centers. These findings conform in large measure to the mammalian and Shaker models. However, virtually all manipulations affecting a highly conserved, central Phe favor the up state of the VSD and the closed KAT1 channel, whereas mutations affecting the electrostatic networks on either side of this Phe favor the down state of the VSD and the open channel. These and additional observations suggest that hydration within the VSD is a major determinant of KAT1 gating.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.