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Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity
Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics
(2006)
Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.
Seasonal, host sex and age-related variations in helminth egg and coccidian oocyst counts were investigated in a naturally infected wild bushbuck (Tragelaphus scriptus) population in Queen Elizabeth National Park, western Uganda from April 2000 to February 2002. The prevalence and mean intensity quantified as the number of eggs and oocysts per gram of faeces were taken as a measure of parasite burdens. Host sex and age-related differences in prevalence values were not found but the overall prevalence of Eimeria sp. was significantly higher during the rainy season, and peak counts were recorded either during or soon after a peak rainfall. A similar trend was observed for Moniezia spp., although the results were marginally not significant. There were also no significant differences in mean intensity values, relative to host sex, age or season.
Bushbuck (Tragelaphus scriptus) often deposit faeces at specific localised defecation sites (LDS). We tested whether LDS have a function in the context of parasite avoidance. In a population of bushbuck in Queen Elizabeth National Park, Uganda, seven radio-collared individuals were observed. We recorded feeding behaviour inside and outside LDS. Furthermore, pasture contamination with gastro-intestinal tract parasites inside and outside LDS was examined. There were significant differences between the expected and the observed feeding rates inside LDS, but, contrary to our prediction, the bushbuck increased their feeding rate inside LDS. There was no significant difference in the parasite contamination of pastures inside and outside LDS. We discuss the hypothesis that LDS mainly serve a social function in bushbuck communities, whereas parasite avoidance seems to play a minor or no role
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).
The photosensitive microvilli of Drosophila photoreceptors R1-R6 are not aligned in parallel over the entire length of the visual cells. In the distal half of each cell, the microvilli are slightly tilted toward one side and, in the proximal half, extremely toward the opposite side. This phenomenon, termed rhabdomere twisting, has been known for several decades, but the developmental and cell biological basis of rhabdomere twisting has not been studied so far. We show that rhabdomere twisting is also manifested as molecular polarization of the visual cell, because phosphotyrosine- containing proteins are selectively partitioned to different sides of the rhabdomere stalk in the distal. and proximal sections of each R1-R6 photoreceptor. Both the asymmetrical segregation of phosphotyrosine proteins and the tilting of the microvilli occur shortly before eclosion of the flies, when eye development in all other aspects is considered to be essentially complete. Establishment of rhabdomere twisting occurs in a light-independent manner, because phosphotyrosine staining is unchanged in dark-reared wild-type flies and in mutants with defects in the phototransduction cascade, ninaE(17) and norpA(P24). We conclude that antiphosphotyrosine immunofluorescence can be used as a light microscopic probe for the analysis of rhabdomere twisting and that microvilli tilting represents a type of planar cell polarity that is established by an active process in the last phase of photoreceptor morphogenesis, just prior to eclosion of the flies.
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by similar to 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold
1. The in situ abundance, biomass and mean cell volume of Actinophrys sol (Sarcodina: Heliozoa), the top predator in an extremely acidic German mining lake (Lake 111; pH 2.65), were determined over three consecutive years (spring to autumn, 2001-03). 2. Actinophrys sol exhibited pronounced temporal and vertical patterns in abundance, biomass and mean cell volume. Increasing from very low spring densities, maxima in abundance and biomass were observed in late June/early July and September. The highest mean abundance recorded during the study was 7 x 10(3) Heliozoa L-1. Heliozoan abundance and biomass were higher in the epilimnion than in the hypolimnion. Actinophrys sol cells from this acidic lake were smaller than individuals of the same species found in other aquatic systems. 3. We determined the growth rate of A. sol using all potential prey items available in, and isolated and cultured from, Lake 111. Prey items included: single-celled and filamentous bacteria of unknown taxonomic affinity, the mixotrophic flagellates Chlamydomonas acidophila and Ochromonas sp., the ciliate Oxytricha sp. and the rotifers Elosa worallii and Cephalodella hoodi. Actinophrys sol fed over a wide-size spectrum from bacteria to metazoans. Positive growth was not supported by all naturally available prey. Actinophrys sol neither increased in cell number (k) nor biomass (k(b)) when starved, with low concentrations of single-celled bacteria or with the alga Ochromonas sp. Positive growth was achieved with single- celled bacteria (k = 0.22 +/- 0.02 d(-1); k(b) = -0.06 +/- 0.02 d(-1)) and filamentous bacteria (k = 0.52 +/- < 0.01 d(- 1); k(b) = 0.66 d(-1)) at concentrations greater than observed in situ, and the alga C. acidophila (up to k = 0.43 +/- 0.03 d(-1); k(b) = 0.44 +/- 0.04 d(-1)), the ciliate Oxytricha sp. (k = 0.34 +/- 0.01 d(-1)) and in mixed cultures containing rotifers and C. acidophila (k = 0.23 +/- 0.02-0.32 +/- 0.02 d(-1); maximum k(b) = 0.42 +/- 0.05 d(-1)). The individual- and biomass-based growth of A. sol was highest when filamentous bacteria were provided. 4. Existing quantitative carbon flux models for the Lake 111 food web can be updated in light of our results. Actinophrys sol are omnivorous predators supported by a mixed diet of filamentous bacteria and C. acidophila in the epilimnion. Heliozoa are important components in the planktonic food webs of 'extreme' environments
The occurrence of two individuals of Zu cristatus at 2 m depth in coastal waters of the Gulf of Castellamare (Tyrrhenian Sea, Italy) together with records of this rare pan-Oceanic mesopelagic species is reported. Analyses of two mitochondrial genes (12 s and 16 s; 936 bp) revealed a 2.6% sequence divergence between Mediterranean and Pacific (Japanese) samples of the species.
Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling.
Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiled-coil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported
Relationships of annelid subtaxa are controversially discussed and additional markers are necessarily needed to get further insights into their evolution. Due to their high content of information, mitochondrial genomes have been proven very useful in phylogenetic analyses. Whereas many complete mitochondrial genomes of arthropods are available, lophotrochozoan taxa are only scarcely represented and this is especially true for annelids. Here we present the complete mitochondrial genome of the orbiniid polychaete Orbinia latreillii. The circular genome is 15,558 bp in size and contains the same 37 genes as found in most other metazoans. As in the case for all studied annelids all genes are transcribed from the same strand. Compared with the known data from other annelids at least five gene translocations must be hypothesized for O. latreillii. A comparison of the available data shows that gene translocations within Annelida seem to be less frequent than in molluscs, but more frequent as previously assumed. Phylogenetic analyses of mitochondrial DNA sequence data and amino acid data support an inclusion Of Sipuncula within Annelida and a closer relationship to orbiniids is recovered for this taxon.
Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird.
G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution. (c) 2006 Elsevier Ltd. All rights reserved.
This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled.
Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. in the blowfly (Calliphora vicina) salivary gland, V- ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the CAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that CAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V, components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus
Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii
(2006)
Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.
The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch syntbase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V-max measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose- synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch
Use of large Acacia trees by the cavity dwelling Black-tailed Tree Rat in the southern Kalahari
(2006)
Recent extensive harvesting of large, often dead Acacia trees in and savanna of southern Africa is cause for concern about the conservation status of the arid savanna and its animal community. We mapped vegetation and nests of the Black-tailed Tree Rat Thallomy's nigricauda to assess the extent to which the rats depend on particular tree species and on the existence of dead, standing trees. The study was conducted in continuous Acacia woodland on the southern and eastern edge of the Kalahari, South Africa. Trees in which there were tree rat nests were compared with trees of similar size and vigour to identify the characteristics of nest sites. Spatial analysis of tree rat distribution was conducted using Ripley's-L function. We found that T nigricauda was able to utilize all available tree species, as long as trees were large and old enough so that cavities were existing inside the stem. The spatial distribution of nest trees did not show clumping at the investigated scale, and we therefore reject the notion of the rats forming colonies when inhabiting continuous woodlands. The selection of a particular tree as a nest site was furthermore depending on the close proximity of the major food plant, Acacia mellifera. This may limit the choice of suitable nest sites. since A. mellifera was less likely to grow within a vegetation patch containing a large trees than in patches without large trees.
It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10-12. Further studies employing the indoleacetic acid-lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [C-14]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains
The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step C-14 labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo- alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed
The ultimate aim of this study is to better understand the relevance of weak electricity in the adaptive radiation of the African mormyrid fish. The chosen model taxon, the genus Campylomormyrus, exhibits a wide diversity of electric organ discharge (EOD) waveform types. Their EOD is age, sex, and species specific and is an important character for discriminating among species that are otherwise cryptic. After having established a complementary set of molecular markers, I examined the radiation of Campylomormyrus by a combined approach of molecular data (sequence data from the mitochondrial cytochrome b and the nuclear S7 ribosomal protein gene, as well as 18 microsatellite loci, especially developed for the genus Campylomormyrus), observation of ontogeny and diversification of EOD waveform, and morphometric analysis of relevant morphological traits. I built up the first convincing phylogenetic hypothesis for the genus Campylomormyrus. Taking advantage of microsatellite data, the identified phylogenetic clades proved to be reproductively isolated biological species. This way I detected at least six species occurring in sympatry near Brazzaville/Kinshasa (Congo Basin). By combining molecular data and EOD analyses, I could show that there are three cryptic species, characterised by their own adult EOD types, hidden under a common juvenile EOD form. In addition, I confirmed that adult male EOD is species-specific and is more different among closely related species than among more distantly related ones. This result and the observation that the EOD changes with maturity suggest its function as a reproductive isolation mechanism. As a result of my morphometric shape analysis, I could assign species types to the identified reproductively isolated groups to produce a sound taxonomy of the group. Besides this, I could also identify morphological traits relevant for the divergences between the identified species. Among them, the variations I found in the shape of the trunk-like snout, suggest the presence of different trophic specializations; therefore, this trait might have been involved in the ecological radiation of the group. In conclusion, I provided a convincing scenario envisioning an adaptive radiation of weakly electric fish triggered by sexual selection via assortative mating due to differences in EOD characteristics, but caused by a divergent selection of morphological traits correlated with the feeding ecology.
For two sympatric species of African weakly electric fish, Campylomormyrus tamandua and Campylomormyrus numenius, we monitored ontogenetic differentiation in electric organ discharge (EOD) and established a molecular phylogeny, based on 2222 bp from cytochrome b, the S7 ribosomal protein gene, and four flanking regions of unlinked microsatellite loci. In C tamandua, there is one common EOD type, regardless of age and sex, whereas in C numenius we were able to identify three different male adult EOD waveform types, which emerged from a single common EOD observed in juveniles. Two of these EOD types formed well supported clades in our phylogenetic analysis. In an independent line of evidence, we were able to affirm the classification into three groups by microsatellite data. The correct assignment and the high pairwise FST values support our hypothesis that these groups are reproductively isolated. We propose that in C numenius there are cryptic species, hidden behind similar and, at least as juveniles, identical morphs.
Es ist bekannt, dass Änderungen im Kohlenstoff- bzw. Stickstoffstaus der Pflanzen zu einer parallelen statt reziproken Änderung der kohlenstoff- und stickstoffhaltigen Primärmetabolite führen. Unter diesem Gesichtspunkt wurden in der vorliegenden Arbeit der Aminosäurestoffwechsel und der Sekundärstoffwechsel unter reduzierten Stickstoffbedingungen untersucht. Zur Beeinflussung des Stickstoffstoffwechsels wurden nitratmangelernährte Tabakwildtyppflanzen und Genotypen mit unterschiedlich stark reduzierter Nitratreduktase-Aktivität verwendet. Dieses experimentelle System erlaubt zusätzlich durch den Vergleich Nitrat defizienter Wildtyppflanzen mit Nitrat akkumulierenden NIA-Transformanten Prozesse zu identifizieren, die durch Nitrat gesteuert werden. Die Analysen der Primär- und Sekundärmetabolite wurde in allen Genotypen diurnal durchgeführt, um auch tageszeitlich abhängige Prozesse zu identifizieren. Die Analyse der absoluten Gehalte aller individuellen Aminosäuren enthüllte bei den meisten erstaunlich stabile diurnale Muster mit einem Anstieg während des Tages und einem Abfall in der Nacht in Wildtyppflanzen gewachsen mit ausreichend Nitrat. Dieses Ergebnis legt die Schlussfolgerung nahe, dass die Biosynthese der Aminosäuren koordiniert abläuft. In Pflanzen mit reduziertem Stickstoffstatus haben diese diurnalen Muster jedoch keinen Bestand. Die Kombination des erzeugten stickstoffbasierten Aminosäuredatensatz in Kombination mit einem bereits erzeugten Aminosäuredatensatz unter kohlenstofflimitierten Bedingungen von Matt et al. (2002) führte durch Hauptkomponentenanalyse (PCA) und Korrelationsanalyse zu dem Ergebnis, dass die Hypothese nach einer koordinierten Aminosäurebiosynthese nicht allgemeine Gültigkeit hat. Die PCA identifizierte Glutamin, Glutamat, Aspartat, Glycin, Pheny-lalanin und Threonin als Faktoren, die den Datensätzen ihre charakteristische Eigenschaft und deren Varianz verleihen. Die Korrelationsanalyse zeigte, dass die sehr guten Korrelationen der individuellen Aminosäuren untereinander in reduzierten Stickstoff- und Kohlenstoffbedingungen sich verschlechtern. Das Verhältnis einer einzelnen Aminosäure relativ zu den anderen führte zur Identifizierung einiger Aminosäuren, die individuelle Antworten auf Stickstoff- und/oder Kohlenstoffstatus zeigen, und/oder speziell auf Nitrat, Licht und/oder den E-nergiestatus der Thylakoidmembran. Glutamat beispielsweise verhält sich in den meisten Situationen stabil, Phenylalanin dagegen zeigt in jeder physiologischen Situation eine individuelle Antwort. Die Ergebnisse dieser Arbeit führen zu einer Erweiterung der Hypothese einer koordinierten Synthese der Aminosäuren dahingehend, dass diese nicht generell für alle Aminosäuren angenommen werden kann. Es gibt einige Aminosäuren deren, Anteile sich situationsbedingt anpassen. Die Reduktion des Stickstoffstatus in nitratmangelernährten Tabakwildtyppflanzen führte zu der, nach der „Carbon-Nutrient-Balance“ Hypothese erwarteten Verlagerung der kohlenstoffreichen Phenylpropanoide und des stickstoffreichen Nikotins. Die Erhöhung der Phenylpropanoidgehalte war nicht in der Nitrat akkumulierenden NIA-Transformante zu beobachten und somit konnte Nitrat als regulatorisches Element identifiziert werden. Ein Einfluss der Vorläufermetabolite konnte ausgeschlossen werden, da sowohl nitratmangelernährter Wildtyp als auch die Nitrat akkumulierende NIA-Transformante ähnliche Gehalte dieser aufwiesen. Genexpressionsanalysen über Mikroarray-Hybridisierung und quantitative RT-PCR zeigten, dass Nitrat durch noch nicht geklärte Mechanismen Einfluss auf die Expression einiger Gene nimmt, die dem Phenylpropanoidstoffwechsels zugeordnet sind. Aus der Arbeit hervorgegangene Veröffentlichungen: Christina Fritz, Natalia Palacios-Rojas, Regina Feil und Mark Stitt (2006) Regulation of Secondary Metabolism by the Carbon-Nitrogen Status in Tobacco: Nitrate Inhibits Large Sectors of Phenylpropanoid Metabolism. Plant Journal 46, 533 - 548 Christina Fritz, Petra Matt, Cathrin Müller, Regina Feil und Mark Stitt (2006) Impact of the Carbon-Nitrogen Status on the Amino Acid Profile in Tobacco Source Leaves. Plant, Cell and Environment 29 (11), 2009 - 2111
Chlamydomonas acidophila, a unicellular green alga, is a dominant phytoplankton species in acidic water bodies, facing severe environmental conditions such as low pH and high heavy metal concentrations. We examined the pH-, and temperature-dependent accumulation of heat-shock proteins in this alga to determine whether heat-shock proteins play a role in adaptation to their environment. Our results show increased heat-shock proteins accumulation at suboptimal pHs, which were not connected with any change in intracellular pH. In comparison to the mesophilic Chlamydomonas reinhardtii, the acidophilic species exhibited significantly higher accumulations of heat-shock proteins under control conditions, indicating an environmental adaptation of increased basal levels of heat-shock proteins. The results suggest that heat- shock proteins might play a role in the adaptation of C. acidophila, and possibly other acidophilic algae, to their extreme environment
More than one century ago, sturgeons were prevalent species in the fish communities of all major German rivers both in the North and the Baltic seas drainages. Since then, the populations declined rapidly due to river damming, overfishing and pollution. The last sturgeon catches in the Baltic drainage system occurred during the late 1960ies. Only a few individual captures have been reported during the last 30 years with the most recent records in the Lake Ladoga ( Russia), where the last confirmed catch was recorded in 1984, and a single individual caught off Estonia in 1996. Today, sturgeons are considered missing or extinct in German waters. First attempts for remediation of the species were undertaken in the mid 1990ies. Subsequently, phylogenetic and population genetic analyses of the species were carried out using mtDNA, microsatellites, and nuclear markers ( SNPs). These genetic analyses using recent and historic material have proven the existence of two different species in the Baltic Sea in what was previously considered to represent the European Atlantic sturgeon only. In the Baltic Sea, the American Atlantic sturgeon ( A. oxyrinchus) succeeded to colonize this brackish water system during the Middle Ages. In the North Sea, the European Atlantic sturgeon ( A. sturio) is considered to be the endemic species. These results led to the separation of the remediation activities in the North Sea and the Baltic Sea tributaries. Further studies on the mechanism that lead to the extinction of A. sturio in Germany and the subsequent succession of the A. oxyrinchus mtDNA haplotype are currently been carried out. Broodstock development using the northernmost populations of A. oxyrinchus is currently under way. As a further prerequisite to re-introduce this species into the Baltic, the evaluation of the status of critical habitats for the early life stages of the American Atlantic sturgeon in the River Odra has been performed in collaboration with the Institute for Inland Fisheries of Poland. Alternative fisheries techniques, based on the data of by-catch of exotic sturgeons in the fishery, are presently developed in close cooperation with the fishery to reduce fisheries related mortality in juvenile sturgeons upon release. Monitoring of habitat utilization and migration characteristics of juvenile fish upon experimental release will have to be carried out shortly, using acoustic telemetry, with the aim to follow the fate of the released fish and to determine the best time-size-release-window for future release programmes.
Sexual dimorphism is expressed as median of the female values in percent of the median of the male values, of 4 length measurements, 3 circumferences, and 5 measurements of corpulence respectively fat. Data were obtained from a cross-sectional sample of more than 41.000 German subjects, aged from birth to age 62. The pattern of sexual dimorphism is similar in the length measurements. Girls are shorter at birth, but they increase in length at higher rates than boys and even temporarily overgrow the boys up to age 12. Thereafter, males show an obvious growth advantage leading to some 6 to 9% more length in adult males. In contrast, female circumferences are always smaller, from birth to senescence. Though, the differences between the sexes are low in circumferences, up to age 13, sexual dimorphism increases to 17% in the thoracic circumference at adulthood. Sexual dimorphism in weight and BMI is comparably with that in length measurements while subcutaneous fat and total body fat content are always higher in females. The results highlight that sexual dimorphism develops at different pace in the various components of the body and that it associates with a sex specific growth tempo.
Simulation models that describe autonomous individual organisms (individual based models, IBM) or agents (agent-based models, ABM) have become a widely used tool, not only in ecology, but also in many other disciplines dealing with complex systems made up of autonomous entities. However, there is no standard protocol for describing such simulation models, which can make them difficult to understand and to duplicate. This paper presents a proposed standard protocol, ODD, for describing IBMs and ABMs, developed and tested by 28 modellers who cover a wide range of fields within ecology. This protocol consists of three blocks (Overview, Design concepts, and Details), which are subdivided into seven elements: Purpose, State variables and scales, Process overview and scheduling, Design concepts, Initialization, Input, and Submodels. We explain which aspects of a model should be described in each element, and we present an example to illustrate the protocol in use. In addition, 19 examples are available in an Online Appendix. We consider ODD as a first step for establishing a more detailed common format of the description of IBMs and ABMs. Once initiated, the protocol will hopefully evolve as it becomes used by a sufficiently large proportion of modellers. (c) 2006 Elsevier B.V. All rights reserved.
Weltweit versuchen Wissenschaftler, künstliche Viren für den Gentransfer zu konstruieren, die nicht reproduktionsfähig sind. Diese sollen die Vorteile der natürlichen Viren besitzen (effizienter Transport von genetischem Material), jedoch keine Antigene auf ihrer Oberfläche tragen, die Immunreaktionen auslösen. Ziel dieses Projektes ist es, einen künstlichen Viruspartikel herzustellen, dessen Basis eine Polyelektrolytenhohlkugel bildet, die mit einer Lipiddoppelschicht bedeckt ist. Um intakte Doppelschichten zu erzeugen, muss die Wechselwirkung zwischen Lipid und Polyelektrolyt (z.B. DNA) verstanden und optimiert werden. Dazu ist es notwendig, die strukturelle Grundlage der Interaktion aufzuklären. Positiv geladene Lipide gehen zwar starke Wechselwirkungen mit der negativ geladenen DNA ein, sie wirken jedoch toxisch auf biologische Zellen. In der vorliegenden Arbeit wurde daher die durch zweiwertige Kationen vermittelte Kopplung von genomischer oder Plasmid-DNA an zwitterionische oder negativ geladene Phospholipide an zwei Modellsystemen untersucht. 1. Modellsystem: Lipidmonoschicht an der Wasser/Luft-Grenzfläche Methoden: Filmwaagentechnik in Kombination mit IR-Spektroskopie (IRRAS), Röntgenreflexion (XR), Röntgendiffraktion (GIXD), Brewsterwinkel-Mikroskopie (BAM), Röntgenfluoreszenz (XRF) und Oberflächenpotentialmessungen Resultate: A) Die Anwesenheit der zweiwertigen Kationen Ba2+, Mg2+, Ca2+ oder Mn2+ in der Subphase hat keinen nachweisbaren Einfluss auf die Struktur der zwitterionischen DMPE- (1,2-Dimyristoyl-phosphatidyl-ethanolamin) Monoschicht. B) In der Subphase gelöste DNA adsorbiert nur in Gegenwart dieser Kationen an der DMPE-Monoschicht. C) Sowohl die Adsorption genomischer Kalbsthymus-DNA als auch der Plasmid-DNA pGL3 bewirkt eine Reduktion des Neigungswinkels der Alkylketten, die auf einen veränderten Platzbedarf der Kopfgruppe zurückzuführen ist. Durch die Umorientierung der Kopfgruppe wird die elektrostatische Wechselwirkung zwischen den positiv geladenen Stickstoffatomen der Lipidkopfgruppen und den negativ geladenen DNA-Phosphaten erhöht. D) Die adsorbierte DNA weist eine geordnete Struktur auf, wenn sie durch Barium-, Magnesium-, Calcium- oder Manganionen komplexiert ist. Der Abstand zwischen parallelen DNA-Strängen hängt dabei von der Größe der DNA-Fragmente sowie von der Art des Kations ab. Die größten Abstände ergeben sich mit Bariumionen, gefolgt von Magnesium- und Calciumionen. Die kleinsten DNA-Abstände werden durch Komplexierung mit Manganionen erhalten. Diese Ionenreihenfolge stellt sich sowohl für genomische DNA als auch für Plasmid-DNA ein. E) Die DNA-Abstände werden durch die Kompression des Lipidfilms nicht beeinflusst. Zwischen der Lipidmonoschicht und der adsorbierten DNA besteht demnach nur eine schwache Wechselwirkung. Offensichtlich befindet sich die durch zweiwertige Kationen komplexierte DNA als weitgehend eigenständige Schicht unter dem Lipidfilm. 2. Modellsystem: Lipiddoppelschicht an der fest/flüssig-Grenzfläche Methoden: Neutronenreflexion (NR) und Quarzmikrowaage (QCM-D) Resultate: A) Das zwitterionische Phospholipid DMPC (1,2-Dimyristoyl-phosphatidylcholin) bildet keine Lipiddoppelschicht auf planaren Polyelektrolytmultischichten aus, deren letzte Lage das positiv geladene PAH (Polyallylamin) ist. B) Hingegen bildet DMPC auf dem negativ geladenen PSS (Polystyrolsulfonat) eine Doppelschicht aus, die jedoch Defekte aufweist. C) Eine Adsorption von genomischer Kalbsthymus-DNA auf dieser Lipidschicht findet nur in Gegenwart von Calciumionen statt. Andere zweiwertige Kationen wurden nicht untersucht. D) Das negativ geladene Phospholipid DLPA (1,2-Dilauryl-phosphatidsäure) bildet auf dem positiv geladenen PAH eine Lipiddoppelschicht aus, die Defekte aufweist. E) DNA adsorbiert ebenfalls erst in Anwesenheit von Calciumionen in der Lösung an die DLPA-Schicht. F) Durch die Zugabe von EDTA (Ethylendiamintetraessigsäure) werden die Calciumionen dem DLPA/DNA-Komplex entzogen, wodurch dieser dissoziiert. Demnach ist die calciuminduzierte Bildung dieser Komplexe reversibel.
The small and large subunits of molybdopterin (MPT) synthase (MOCS2A and MOCS2B), are both encoded by the MOCS2 gene in overlapping and shifted open reading frames (ORFs), which is a highly unusual structure for eukaryotes. Theoretical analysis of genomic sequences suggested that the expression of these overlapping ORFs is facilitated by the use of alternate first exons leading to alternative transcripts. Here, we confirm the existence of these overlapping transcripts experimentally. Further, we identified a deletion in a molybdenum cofactor deficient patient, which removes the start codon for the small subunit (MOCS2A). We observed undisturbed production of both transcripts, while Western blot analysis demonstrated that MOCS2B, the large subunit, is unstable in the absence of MOCS2A. This reveals new insights into the expression of this evolutionary ancient anabolic system.
The binding of acetylcholinesterase (AChE) to a propidium-modified piezoelectric quartz crystal and its surface enzymatic activity have been investigated. Propidium binds to a site remote to the active center of AChE - the peripheral anionic site (PAS) - which is located on the rim of the gorge to the active site. The gold electrodes of the quartz crystal were first modified with 11-mercaptoundecanoic acid to which propidium was coupled. AChE binding was monitored by a quartz crystal nanobalance (QCN), followed by amperometric activity evaluation of the AChE loaded on the sensor. Interestingly, the binding is strong but does not inhibit AChE. However, an excess of propidium in solution inhibits the immobilized enzyme. The surface enzymatic activities observed depend on the amount of enzyme and differ according to the type and species, i.e. number of enzyme subunits (Electrophorus electricus tetrameric, Drosophila melanogaster mono- and dimeric form - DmAChE). The operational stability and regeneration, effect of propidium in solution and detection limit for substrate for various AChEs were investigated amperometrically.
We summarize here the development of various piezoelectric biosensors utilizing cholinesterase (ChE) as the recognition element. In our work we studied the interaction between cholinesterase and its ligands (propidium, carnitine, benzylgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) and paraoxon). The sensor modification was based on a self-assembled monolayer (SAM) of a thiol compound (11-mercaptoundecanoic acid) on the gold electrode and the subsequent covalent coupling of the cholinesterase ligand to this SAM. The ligand-modified piezoelectric sensors were placed in a flow system to allow the on-line monitoring of cholinesterase binding and the enzymatic activity quantification by amperometry. Cholinesterases from different species-acetylcholinesterase (AChE) from Electrophorus electricus , AChE from Drosophila melanogaster , and butyrylcholinesterase (BChE) of human origin-were tested on the various immobilized ligands. Our research allowed the development of a competitive assay for the detection of organophosphates in river water samples using the BZE-DADOO-modified piezosensor. Another direction of research was pointed on the characterization of the interactions between ChE and its ligands. The kinetic binding constants were derived using a one- to-one binding model
Maldanids are usually divided into several subfamilies: Euclymeninae, Lumbriclymeninae, Maldaninae, Nicomachinae, Rhodininae, Clymenurinae, Notoproctinae, and Boguinae. The taxonomy of maldanids and the delimination of these taxa are mainly based on head morphology, total number of segments, chaetal structure, shape of the pygidium, and position of the anus. The maldanid ingroup relationships, as well as the monophyly of the proposed subfamilies, have so far not been investigated. Pilgrim (1977) described a shift of the notopodial chaetal rows from a transverse direction in anterior chaetigers to a more longitudinal one in posterior chaetigers in Clymene torquata (Leidy, 1855) and Euclymene oerstedii (Claparede, 1863), both belonging to the Euclymeninae. We investigated several maldanid species to assess the usefulness of this character for maldanid systematics and used 3D-reconstruction techniques to reveal the complete geometry of the chaetal sacs. Our investigation shows that a shift is apparent in Euclymene, Axiothella, Johnstonia (all Euclymeninae) and Clymenura (Clymenurinae), but absent in species like Maldane sarsi (Malmgren, 1865), Metasychis disparidentata (Moore, 1904) (both Maldaninae) and Petaloproctus borealis Ardwisson, 1906 (Nicomachinae). The shift is not typical for sedentary polychaetes and is apomorphic within maldanid polychaetes. It thus argues for a close relationship between Euclymeninae and Clymenurinae. The investigation of further maldanid species of different subfamilies may shed additional light on maldanid systematics.
G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution.
Die vorliegende Arbeit wurde im Zeitraum von Oktober 2002 bis November 2005 an dem Institut für Biochemie und Biologie der Universität Potsdam in Kooperation mit dem Institut für Chemie des GKSS Forschungszentrums in Teltow unter der Leitung von Herrn Prof. Dr. B. Micheel und Herrn Prof. Dr. Th. Groth angefertigt. Im Rahmen dieser Arbeit wurden die Wechselwirkungen von Immunzellen mit verschiedenen Kultursubstraten untersucht. Dafür wurden drei verschiedene Hybridomzelllinien eingesetzt. Eine Hybridomzelllinie (K2) ist im Laufe dieser Arbeit hergestellt und etabliert worden. Der Einsatz von synthetischen und proteinbeschichteten Kulturoberflächen führte bei Hybridomzellen zu einer deutlich gesteigerten Antikörpersynthese im Vergleich zu herkömmlichen Zellkulturmaterialien. Obwohl diese Zellen in der Regel als Suspensionszellen kultiviert werden, führten die eingesetzten Polymermembranen (PAN, NVP) zu einer verbesserten Antikörpersynthese (um 30%) gegenüber Polystyrol als Referenz. Es konnte gezeigt werden, dass es einen Zusammenhang zwischen der Produktivität und dem Adh asionsverhalten der Hybridomzellen gibt. Um den Einfluss von Proteinen der extrazellulären Matrix auf Zellwachstum und Antikörpersynthese von Hybridomzellen zu untersuchen, wurden proteinbeschichtete Polystyrol-Oberflächen eingesetzt. Für die Modifikationen wurden Fibronektin, Kollagen I, Laminin und BSA ausgewählt. Die Modifikation der Polystyrol-Oberfläche mit geringen Mengen Fibronektin (0,2-0,4 µg/ml) führte zu einer beträchtlichen Steigerung der Antikörpersynthese um 70-120%. Für Kollagen I- und BSA-Beschichtungen konnten Steigerungen von 40% beobachtet werden. Modifikationen der Polystyrol-Oberfläche mit Laminin zeigten nur marginale Effekte. Durch weitere Versuche wurde bestätigt, dass die Adhäsion der Zellen an Kollagen I- und Laminin-beschichteten Oberflächen verringert ist. Die alpha2-Kette des alpha2beta1-Integrins konnte auf der Zelloberfläche nicht nachgewiesen werden. Durch ihr Fehlen wird wahrscheinlich die Bindungsfähigkeit der Zellen an Kollagen I und Laminin beeinflusst. Durch die Ergebnisse konnte gezeigt werden, dass Hybridomzellen nicht nur Suspensionszellen sind und das Kultursubstrate das Zellwachstum und die Produktivität dieser Zellen stark beeinflussen können. Der Einsatz von synthetischen und proteinbeschichteten Kultursubstraten zur Steigerung der Antikörpersynthese kann damit für die industrielle Anwendung von großer Relevanz sein. Für die Modellierung einer Lymphknotenmatrix wurden Fibronektin, Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose in verschiedenen Kombinationen an Glasoberflächen adsorbiert und für Versuche zur In-vitro-Immunisierung eingesetzt. Es konnte gezeigt werden, dass die Modifikation der Oberflächen die Aktivierung und Interaktion von dendritischen Zellen, T- und B-Lymphozyten begünstigt, was durch den Nachweis spezifischer Interleukine (IL12, IL6) und durch die Synthese spezifischer Antikörper bestätigt wurde. Eine spezifische Immunreaktion gegen das Antigen Ovalbumin konnte mit den eingesetzten Zellpopulationen aus Ovalbumin-T-Zell-Rezeptor-transgenen Mäusen nachgewiesen werden. Die In-vitro-Immunantwort wurde dabei am stärksten durch eine Kombination von Kollagen I, Heparansulfat und N-Acetylglucosamin-mannose auf einer Glasoberfläche gefördert. Die Etablierung einer künstlichen Immunreaktion kann eine gesteuerte Aktivierung bzw. Inaktivierung von körpereigenen dendritischen Zellen gegen bestehende Krankheitsmerkmale in vitro ermöglichen. Durch die Versuche wurden Grundlagen für spezifische Immunantworten erarbeitet, die u.a. für die Herstellung von humanen Antikörpern eingesetzt werden können.
Soil seed banks near rubbing trees indicate dispersal of plant species into forests by wild boar
(2006)
Current knowledge about processes that generate long-distance dispersal of plants is still limited despite its importance for persistence of populations and colonization of new potential habitats. Today wild Large mammals are presumed to be important vectors for long-distance transport of diaspores within and between European temperate forest patches, and in particular wild boars recently came into focus. Here we use a specific habit of wild boar, i.e. wallowing in mud and subsequent rubbing against trees, to evaluate epizoochorous dispersal of vascular plant diaspores. We present soil seed bank data from 27 rubbing trees versus 27 control trees from seven forest areas in Germany. The mean number of viable seeds and the plant species number were higher in soil samples near rubbing trees compared with control trees. Ten of the 20 most frequent species were more frequent, and many species exclusively appeared in the soil samples near rubbing trees. The large number of plant species and seeds - more than 1000 per tree - in the soils near rubbing trees is difficult to explain unless the majority were dispersed by wild boar. Hooked and bristly diaspores, i.e. those adapted to epizoochory, were more frequent; however, many species with unspecialized diaspores occurred exclusively near rubbing trees. As opposed to plant species closely tied to forests species which occur both in forest and open vegetation and non-forest species were more frequent near rubbing trees compared with controls. These findings are consistent with previous studies on diaspore loads in the coats and hooves of shot wild boars. However, our method allows to identify the transport of diaspores from the open landscape into forest stands, where they might especially emerge after disturbance, and a clustered distribution of epizoochorically dispersed seeds. Moreover, accumulation of seeds of wetness indicators near rubbing trees demonstrates directed dispersal of plant species inhabiting wet places among remote wallows.
Soil seed banks near rubbing trees indicate dispersal of plant species into forests by wild boar
(2006)
Current knowledge about processes that generate long-distance dispersal of plants is still limited despite its importance for persistence of populations and colonization of new potential habitats. Today wild large mammals are presumed to be important vectors for long-distance transport of diaspores within and between European temperate forest patches, and in particular wild boars recently came into focus. Here we use a specific habit of wild boar, i.e. wallowing in mud and subsequent rubbing against trees, to evaluate epizoic dispersal of vascular plant diaspores. We present soil seed bank data from 27 rubbing trees versus 27 control trees from seven forest areas in Germany. The mean number of viable seeds and the plant species number were higher in soil samples near rubbing trees compared with control trees. Ten of the 20 most frequent species were more frequent, and many species exclusively appeared in the soil samples near rubbing trees. The large number of plant species and seeds – approximated > 1000 per tree – in the soils near rubbing trees is difficult to explain unless the majority were dispersed by wild boar. Hooked and bristly diaspores, i.e. those adapted to epizoochory, were more frequent, above that many species with unspecialised diaspores occurred exclusively near rubbing trees. Different to plant species closely tied to forest species which occur both in forest and open vegetation, and non-forest species were more frequent near rubbing trees compared with controls. These findings are consistent with previous studies on diaspore loads in the coats and hooves of shot wild boars. However, our method allows to identify the transport of diaspores from the open landscape into forest stands where they might especially emerge after disturbance, and a clustered distribution of epizoochorically dispersed seeds. Moreover, accumulation of seeds of wetness indicators near rubbing trees demonstrates directed dispersal of plant species inhabiting wet places between remote wallows.
The late-Quaternary climate history of monsoonal Central Asia was inferred from 75 palaeoclimatic records which provide information about moisture conditions in the last 50 ka (or part of this period). Wet conditions occurred during middle and late Marine Isotope Stage 3, while the Last Glacial Maximum (LGM) was characterized by dry climate conditions in the region. A stepwise climate amelioration is suggested by the climate records following the LGM. Several climate signals of this period, which were reported from high-latitude ice core records, are preserved in archives from monsoonal Central Asia as well. During the early Holocene, high effective moisture was inferred from most records from the area dominated by the Indian Monsoon (e.g. the Tibetan Plateau) suggesting that Holocene optimal climate conditions occurred there during this period. In contrast, areas which are dominated by the South-East Asian monsoon (SE Monsoon) and the Westerlies (in north-western and north-central China, Mongolia) do not uniformly show an early Holocene climate optimum. For this area optimal conditions prevailed during the mid-Holocene. These apparent contradictions can possibly be explained by the regional uplift and descent of air masses in the Holocene. During the early Holocene, strengthened insolation possibly caused an enhanced low-level convergence over the Tibetan Plateau which led to the intensification of the summer monsoon. The strong air uplift caused intensified precipitation and air divergence in the upper troposphere over the Tibetan Plateau. The areas adjacent to the north therefore experienced an intensified descent of air masses and consequently increased aridity. The majority of the palaeoclimatic records suggest reduced effective moisture since the late Holocene in the region.
Recent and subfossil pollen spectra from the Alashan Plateau are presented in order to provide information on desert plant representation and on recent changes in vegetation and climate in this remote area in northern China. The desert vegetation composition is faithfully represented by the surface pollen spectra. The comparison of the desert plant species to the related pollen taxa yielded the following sequence from over-representation to under- representation: Chenopodiaceae, Artemisia, Ephedra fragilis-type s.l., Reaumuria, Nitraria and Calligonum. A 72 cm long sediment record from a small hydrologically-closed inter-dune lake (SE Badan Jilin Sand Sea, southern Alashan Plateau) covering the past similar to 160 years (dated by(137)Cs) was analysed palynologically. Intervals of denser Artemisia coverage on the sand dunes around the lake, indicating wetter climate, occurred from the mid-1850s to the mid-1870s, during the first two decades of the 20th century and from the late 1930s to the beginning of the 1960s
A 13.94-m-long sediment core, collected from a medium-sized lake in the Qilian Mountains (NE Tibetan Plateau, China), was analysed palynologically at 81 horizons. The interpretation of indicator taxa yielded various vertical shifts of the vegetation belts. These palaeovegetation results have been checked with lake surface pollen spectra from 8 takes representing different altitudinal vegetation belts. Our main findings are the following: A short period of the late Marine Isotope Stage 3 (around similar to 46,000 yr ago) was characterized by interglacial temperature conditions with a tree line above its present-day altitude. During the LGM, the vicinity of the lake was not covered by ice but by sparse alpine vegetation and alpine deserts, indicating that the climate was colder by similar to 4-7 degrees C than today Markedly higher temperatures were inferred from higher arboreal pollen frequencies between similar to 13,000 and similar to 7000 yr ago with a Holocene temperature optimum and a maximal Picea-Betula mixed-forest expansion between similar to 9000 and similar to 7000 yr ago, when temperatures exceeded the present-day conditions by at least 1-2 degrees C. Alpine steppes and meadows and sub-alpine shrub vegetation dominated around the lake since the middle Holocene, suggesting that vegetation and climate conditions were exceptionally stable in comparison to previous periods.
Charakterisierung von Transportmechanismen in der Speicheldrüse der Schabe Periplaneta americana
(2006)
Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe Periplaneta americana durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt. Mikrofluorometrische Ca<sup>2+-, Na<sup>+- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. Wesentliche Ergebnisse dieser Arbeit waren: <ul> <li>Messungen des intrazellulären pH (pHi) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pHi-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na<sup>+-K<sup>+-2Cl<sup>--Symporter und der Cl<sup>--HCO3<sup>--Antiporter, gekoppelt mit dem Na<sup>+ H<sup>+-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO2/HCO3<sup>--Verfügbarkeit ab und war Ca<sup>2+-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H<sup>+-ATPase), aber von der Aktivität der basolateralen Na<sup>+-K<sup>+-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H<sup>+-ATPase und Na<sup>+-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH4Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pHi in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na<sup>+]i und, zeitlich verzögert, auch der [Ca<sup>2+]i. Der [Na<sup>+]i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca<sup>2+]i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na<sup>+]i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na<sup>+-Ca<sup>2+-Antiporter in den Ca<sup>2+-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca<sup>2+]i-Erhöhung könnte an der Regulation der Na<sup>+-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na<sup>+-K<sup>+-ATPase noch die V-H<sup>+-ATPase waren an der Resorption beteiligt.</li> </ul> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich.
Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca2+ concentration ([Ca2+](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca2+ influx from the extracellular space. Additionally, dopamine induces a massive [Na+](i) elevation via the Na+- K+-2Cl(-) cotransporter (NKCC). We have reasoned that Ca2+-entry is mediated by the Na+-Ca2+ exchanger (NCE) operating in the Ca2+-entry mode. To test this hypothesis, [Ca2+](i) and [Na+](i) were measured by using the fluorescent dyes Fura- 2, Fluo-3, and SBFI. Inhibition of Na+-entry from the extracellular space by removal of extracellular Na+ or inhibition of the NKCC by 10 mu M bumetanide did not influence resting [Ca2+]i but completely abolished the dopamine-induced [Ca2+](i) elevation. Simultaneous recordings of [Ca2+](i) and [Na+](i) revealed that the dopamine-induced [Na+](i) elevation preceded the [Ca2+](i) elevation. During dopamine stimulation, the generation of an outward Na+ concentration gradient by removal of extracellular Na+ boosted the [Ca2+](i) elevation. Furthermore, prolonging the dopamine-induced [Na+](i) rise by blocking the Na+/K+-ATPase reduced the recovery from [Ca2+](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na+](i), which reverses the NCE activity into the reverse mode causing a graded [Ca2+](i) elevation in the duct cells.
Wild birds are considered a potential reservoir or a carrier of viral diseases and may therefore play a role in the epidemiology of economically important or zoonotic diseases. In 2001 and 2002, a survey with special emphasis oil virus isolation in migrating waders and some other birds were conducted. In one of the most important inland resting sites for migratory waterfowl, tracheal and cloacal swabs were collected from 465 waders representing 19 different species, and 165 other birds that were not captured on purpose. A total of 42 avian viruses were isolated, 34 of these were identified as paramyxoviruses (PMVs). The majority of isolates came from waders and wild ducks, and were characterized as PMV-1. In contrast, PMV-4 was found in wild ducks only, PMV-6 was mainly detected in wader species. Four avian influenza viruses (ATVs), belonging to H4 and H3 haemagglutinin subtype, were isolated from wild duck species. Furthermore, four reo-like viruses were isolated from one particular wader species for the first time. The majority of virus positive birds were < 1 year old and did not show any clinical symptoms. There was no evidence for the presence of West Nile virus in these birds. These results confirm that the restricted resting sites in Western Europe must be considered as important locations for the intra- and interspecies transmission of avian viruses