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Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His(6)) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis
Xin is a protein that is expressed during early developmental stages of cardiac and skeletal muscles. Immunolocalization studies indicated a peripheral localization in embryonic mouse heart, where Xin localizes with beta- catenin and N-cadherin. In adult tissues, Xin is found primarily in the intercalated discs of cardiomyocytes and the myotendinous junctions of skeletal muscle cells, both specialized attachment sites of the myofibrillar ends to the sarcolemma. A large part of the Xin protein consists of unique 16 amino acid repeats with unknown function. We have investigated the characteristics of the Xin repeats by transfection experiments and actin-binding assays and ascertained that, upon expression in cultured cells, these repeats bind to and stabilize the actin-based cytoskeleton. In vitro co- sedimentation assays with skeletal muscle actin indicated that they not only directly bind actin filaments, but also have the capability of arranging microfilaments into networks that sediment upon low-speed centrifugation. Very similar repeats were also found in Xin-repeat protein 2' (XIRP2), a novel protein that seems to be expressed mainly in striated muscles. Human XIRP2 contains 28 Xin repeats with properties identical to those of Xin. We conclude that the Xin repeats define a novel, repetitive actin-binding motif present in at least two different muscle proteins. These Xin- repeat proteins therefore constitute the first two members of a novel family of actin-binding proteins
Vom Seidenbau in Krausnick
(2004)
Herbivore populations are commonly restricted by resource limitation, by predation or a combination of the two. Food supplement experiments are suitable for investigating the extent of food limitation at any given time. The main part of this study was performed in an extremely acidic lake (pH 2.7) where the food web consists of only a few components and potential food sources for herbivores are restricted to two flagellates. Life table experiments proved that Chlamydomonas was a suitable food source whereas Ochromonas was an unsuitable food source. The two flagellates and the two rotifers exhibit a pronounced vertical distribution pattern. In this study, a series of food supplement experiments were performed in order to: (1) quantify and compare potential resource limitation of two primary consumers (Cephalodella hoodi and Elosa worallii, Rotatoria) over time, (2) compare their response at different temperatures, (3) evaluate the effect of having an unsuitable food source alongside a valuable one, (4) estimate the effect of predation on rotifers by Heliozoa, and (5) compare the results with those from other acidic lakes. Additionally, the spatio- temporal population dynamics of both species were observed. The field data confirmed a vertical separation of the two species with E. worallii dominating in the upper water layers, and C. hoodi in the deeper, cooler water layers. The results from the food supplement experiments in which Chlamydomonas served as the supplemented suitable food source showed that the two rotifers were food limited in the epilimnion throughout the season to different extents, with Cephalodella being more severely food limited than Elosa. The experiments at different temperatures provided evidence that Elosa had a higher optimum temperature for growth than Cephalodella. When the unsuitable food algae Ochromonas was added alongside the suitable food source Chlamydomonas, C. hoodi was unaffected but E. worallii was negatively affected. Predation of Heliozoa on rotifers was observed but the total effect on the rotifer dynamics is probably low. The comparison with other lakes showed that resource limitation also occurred in one other lake, although to a lesser extent. Overall, the vertical separation of the two rotifers could be explained by both their differential extent of resource limitation and differential response to temperature.
Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics
Variation in nitrogen deposition and available soil nitrogen in a forest–grassland ecotone in Canada
(2004)
Regional variation in nitrogen (N) deposition increases plant productivity and decreases species diversity, but landscape- or local-scale influences on N deposition are less well-known. Using ion-exchange resin, we measured variation of N deposition and soil N availability within Elk Island National Park in the ecotone between grassland and boreal forest in western Canada. The park receives regionally high amounts of atmospheric N deposition (22 kg ha⁻¹ yr⁻¹). N deposition was on average higher ton clayrich luvisols than on brunisols, and areas burned 1 – 15 years previously received more atmospheric N than unburned sites. We suggest that the effects of previous fires and soil type on deposition rate act through differences in canopy structure. The magnitude of these effects varied with the presence of ungulate grazers (bison, moose, elk) and vegetation type (forest, shrubland, grassland). Available soil N (ammonium and nitrate) was higher in burned than unburned sites in the absence of grazing, suggesting an effect of deposition. On grazed sites, differences between fire treatments were small, presumably because the removal of biomass by grazers reduced the effect of fire. Aspen invades native grassland in this region, and our results suggest that fire without grazing might reinforce the expansion of forest into grassland facilitated by N deposition.
Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the “hmmer” program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes.
Transcription factors, growth factors and signal cascades capable of priming morphogenesis of heart
(2004)
Cytochrome c was immobilized on screen-printed thick-film gold electrodes by a self-assembly approach using mixed monolayers of mercaptoundecanoic acid and mercaptoundecanol. Cyclic voltammetry revealed quasi-reversible electrochemical behavior of the covalently fixed protein with a formal potential of +10 mV vs. Ag/AgCl. Polarized at +150 mV vs. Ag/AgCl the electrode was found to be sensitive to superoxide radicals in the range 300-1200 nmol L-1. Compared with metal needle electrodes sensitivity and reproducibility could be improved and combined with the easiness of preparation. This allows the fabrication of disposable sensors for nanomolar superoxide concentrations. By changing the electrode potential the sensor can be switched from response to superoxide radicals to hydrogen peroxide-another reactive oxygen species. H2O2 sensitivity can be provided in the range 10-1000 mumol L-1 which makes the electrode suitable for oxidative stress studies
The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase- activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia ( brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non- proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease
The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II
The acinar salivary glands of the cockroach, Periplaneta americana, are innervated by dopaminergic and serotonergic nerve fibers. Serotonin stimulates the secretion of protein-rich saliva, whereas dopamine causes the production of protein-free saliva. This suggests that dopamine acts selectively on ion-transporting peripheral cells within the acini and the duct cells, and that serotonin acts on the protein-producing central cells of the acini. We have investigated the pharmacology of the dopamine-induced secretory activity of the salivary gland of Periplaneta americana by testing several dopamine receptor agonists and antagonists. The effects of dopamine can be mimicked by the non-selective dopamine receptor agonist 6,7-ADTN and, less effectively, by the vertebrate D1 receptor-selective agonist chloro-APB. The vertebrate D1 receptor-selective agonist SKF 38393 and vertebrate D2 receptor-selective agonist R(-)- TNPA were ineffective. R(+)-Lisuride induces a secretory response with a slower onset and a lower maximal response compared with dopamine-induced secretion. However, lisuride-stimulated glands continue secreting saliva, even after lisuride-washout. Dopamine-induced secretions can be blocked by the vertebrate dopamine receptor antagonists cis(Z)- flupenthixol, chlorpromazine, and S(+)-butaclamol. Our pharmacological data do not unequivocally indicate whether the dopamine receptors on the Periplaneta salivary glands belong to the D1 or D2 subfamily of dopamine receptors, but we can confirm that the pharmacology of invertebrate dopamine receptors is remarkably different from that of their vertebrate counterparts. (C) 2004 Elsevier Ltd. All rights reserved
The development of the cardiovascular system in embryoid bodies deriverd from embryonic stem cells
(2004)
Temporary-pond species can be expected to use environmental cues to predict the onset of adverse conditions, while permanent-pond species may be insensitive to such cues. Temperature is such a potential cue in temporary waterbodies, as if fluctuates more widely with decreasing pond size than in deeper permanent ponds. We compared the temperature-induced response of a permanent-pond and a temporary-pond cyclopoid copepod focusing on juvenile development duration, diapause induction and survival during diapause. Nonlinear regression analysis suggested a stronger effect of temperature on the duration of juvenile development in the temporary-pond species. This species also showed a higher and temperature-dependent variation in development duration (highest coefficient of variation 26%) compared with the permanent species, for which variation was lower and similar at all temperatures (maximal coefficient of variation 6%). Temperature significantly influenced the induction of diapause in the temporary-pond species, where the percentage of individuals entering diapause increased from 0% at 5degreesC and 10degreesC to 63% at 15degreesC and 91% at 20degreesC. In the permanent-pond species, diapause induction was independent of temperature and was induced in 100% of experimental specimens. This suggests an obligatory diapause in the permanent-pond species, a type of dormancy that has not been described previously for cyclopoid copepods. Survival during diapause in both species was higher when the diapausing copepodid stage was reached at lower temperatures. At higher temperatures, the temporary-pond species survived longer than the permanent-pond species. These results suggest different temperature optima of the two species. The strategy displayed by the permanent-pond species might be selected for in more stable habitats and may preclude the colonization of temporary ponds. Higher flexibility in life-history traits and the use of temperature as an environmental cue in the temporary-pond species could be favoured in unpredictable habitats
In this work different approaches are undertaken to improve the understanding of the sucrose-to-starch pathway in developing potato tubers. At first an inducible gene expression system from fungal origin is optimised for the use of studying metabolism in the potato tuber. It is found that the alc system from Aspergillus nidulans responds more rapidly to acetaldehyde than ethanol, and that acetaldehyde has less side-effects on metabolism. The optimal induction conditions then are used to study the effects of temporally controlled cytosolic expression of a yeast invertase on metabolism of potato tubers. The observed differences between induced and constitutive expression of the invertase lead to the conclusion that glycolysis is induced after an ATP demand has been created by an increase in sucrose cycling. Furthermore, the data suggest that in the potato tuber maltose is a product of glucose condensation rather than starch degradation. In the second part of the work it is shown that the expression of a yeast invertase in the vacuole of potato tubers has similar effects on metabolism than the expression of the same enzyme in the apoplast. These observations give further evidence to the presence of a mechanism by which sucrose is taken up via endocytosis to the vacuole rather than via transporters directly to the cytosol. Finally, a kinetic in silico model of sucrose breakdown is presented that is able to simulate this part of potato tuber metabolism on a quantitative level. Furthermore, it can predict the metabolic effects of the introduction of a yeast invertase in the cytosol of potato tubers with an astonishing precision. In summary, these data prove that inducible gene expression and kinetic computer models of metabolic pathways are useful tools to greatly improve the understanding of plant metabolism.
Im Mittelpunkt dieser Arbeit standen Signaltransduktionsprozesse in den Strukturen der Kraftübertragung quergestreifter Muskelzellen, d. h. in den Costameren (Zell-Matrix-Kontakten) und den Glanzstreifen (Zell-Zell-Kontakten der Kardiomyozyten).Es ließ sich zeigen, dass sich die Morphologie der Zell-Matrix-Kontakte während der Differenzierung von Skelettmuskelzellen dramatisch ändert, was mit einer veränderten Proteinzusammensetzung einhergeht. Immunfluoreszenz-Analysen von Skelettmuskelzellen verschiedener Differenzierungsstadien implizieren, dass die Signalwege, welche die Dynamik der Fokalkontakte in Nichtmuskelzellen bestimmen, nur für frühe Stadien der Muskeldifferenzierung Relevanz haben können. Ausgehend von diesem Befund wurde begonnen, noch unbekannte Signalwege zu identifizieren, welche die Ausbildung von Costameren kontrollieren: In den Vorläuferstrukturen der Costamere gelang es, eine transiente Interaktion der Proteine Paxillin und Ponsin zu identifizieren. Biochemische Untersuchungen legen nahe, dass Ponsin über eine Skelettmuskel-spezifische Insertion im Carboxyterminus das Adapterprotein Nck2 in diesen Komplex rekrutiert. Es wird vorgeschlagen, dass die drei Proteine einen ternären Signalkomplex bilden, der die Umbauvorgänge der Zell-Matrix-Kontakte kontrolliert und dessen Aktivität von mitogen activated protein kinases (MAPK) reguliert wird.Die Anpassungsvorgänge der Strukturen der Kraftübertragung an pathologische Situtation (Kardiomyopathien) in der adulten quergestreiften Muskulatur wurden ausgehend von einem zweiten Protein, dem muscle LIM protein (MLP), untersucht. Es konnte gezeigt werden, dass ein mutiertes MLP-Protein, das im Menschen eine hypertrophe Kardiomyopathie (HCM) auslöst, strukturelle Defekte aufweist und weniger stabil ist. Weiterhin zeigte dieses mutierte Protein eine verringerte Bindungsfähigkeit an die beiden Liganden N-RAP und alpha-Actinin. Die molekulare Grundlage der HCM-verursachenden Mutationen im MLP-Gen könnte folglich eine Veränderung der Homöostase im ternären Komplex MLP – N-RAP – alpha-Actinin sein. Die Expressionsdaten eines neu generierten monoklonalen MLP-Antikörpers deuten darauf hin, dass die Funktionen des MLP nicht nur für die Integrität des Myokards, sondern auch für die der Skelettmuskulatur notwendig sind.
Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D140XD142XE144 sequence motif In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue. (C) 2003 Elsevier B.V. All rights reserved
Structure and reactivity of a biological soil crust from a xeric sandy soil in Central Europe
(2004)
The investigation was designed to explore the structure, composition and activity of a biological soil crust on an acidic, sandy soil from a temperate climate. The crust covers several hundreds of square meters on the hilltop of a large terminal moraine. The conjugate alga Zygogonium ericetorum forms the essential matrix for the crust, a dense web of algal filaments with interspersed lichens and mosses. The crust is composed of three layers, with an uppermost layer consisting nearly entirely of a dense algal mat. In lower layers, a parasitic fungus, penetrating the algal cells, is another important component of the crust community. In this soil crust, photosynthetic and respiratory activity is stabilized at low water activities.
Structural and animal species diversity in arid and semi-arid savannas of the southern Kalahari
(2004)
Vertical differences in food web structure were examined in an extremely acidic, iron-rich mining lake in Germany (Lake 111; pH 2.6, total Fe 150mg L-1) during the period of stratification. We tested whether or not the seasonal variation of the plankton composition is less pronounced than the differences observed over depth. The lake was strongly stratified in summer, and concentrations of dissolved organic carbon and inorganic carbon were consistently low in the epilimnion but high in the hypolimnion. Oxygen concentrations declined in the hypolimnion but were always above 2mg L-1. Light attenuation did not change over depth and time and was governed by dissolved ferric iron. The plankton consisted mainly of single-celled and filamentous bacteria, the two mixotrophic flagellates Chlamydomonas sp. and Ochromonas sp., the two rotifer species Elosa worallii and Cephalodella hoodi, and Heliozoa as top predators. We observed very few ciliates and rhizopods, and no heterotrophic flagellates, crustaceans or fish. Ochromonas sp., bacterial filaments, Elosa and Heliozoa dominated in the epilimnion whereas Chlamydomonas sp., single-celled bacteria and Cephalodella dominated in the hypolimnion. Single-celled bacteria were controlled by Ochromonas sp. whereas the lack of large consumers favoured a high proportion of bacterial filaments. The primarily phototrophic Chlamydomas sp. was limited by light and CO2 and may have been reduced due to grazing by Ochromonas sp. in the epilimnion. The distribution of the primarily phagotrophic Ochromonas sp. and of the animals seemed to be controlled by prey availability. Differences in the plankton composition were much higher between the epilimnion and hypolimnion than within a particular stratum over time. The food web in Lake 111 was extremely species-poor enabling no functional redundancy. This was attributed to the direct exclusion of species by the harsh environmental conditions and presumably enforced by competitive exclusion. The latter was promoted by the low diversity at the first trophic level which, in turn, was attributed to relatively stable growth conditions and the independence of resource availability (inorganic carbon and light) from algal density. Ecological theory suggests that low functional redundancy promotes low stability in ecosystem processes which was not supported by our data.
We have identified a gene, ST18 (suppression of tumorigenicity 18, breast carcinoma, zinc-finger protein), within a frequent imbalanced region of chromosome 8q11 as a breast cancer tumor suppressor gene. The ST18 gene encodes a zinc-finger DNA-binding protein with six fingers of the C2HC type (configuration Cys-X5-Cys-X12-His-X4-Cys) and an SMC domain. ST18 has the potential to act as transcriptional regulator. ST18 is expressed in a number of normal tissues including mammary epithelial cells although the level of expression is quite low. In breast cancer cell lines and the majority of primary breast tumors, ST18 mRNA is significantly downregulated. A 160 bp region within the promoter of the ST18 gene is hypermethylated in about 80% of the breast cancer samples and in the majority of breast cancer cell lines. The strong correlation between ST18 promoter hypermethylation and loss of ST18 expression in tumor cells suggests that this epigenetic mechanism is responsible for tumor-specific downregulation. We further show that ectopic ST18 expression in MCF-7 breast cancer cells strongly inhibits colony formation in soft agar and the formation of tumors in a xenograft mouse model
The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4'-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4'-dithiodipyridin and dithionite modified electrodes. A formal potential (E-0') of -373 mV vs Ag/AgCl 1 M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis. (C) 2003 Elsevier Inc. All rights reserved
A nutrient enrichment experiment was conducted in order to study the role of nitrogen (N), phosphorus (P) and the N:P ratio on the early summer phytoplankton community in the Archipelago Sea, northern Baltic Sea. The phytoplankton community was, in terms of chlorophyll a and total biomass, primarily N-limited, but the individual species varied in their responses to the nutrient supply. The recorded overall N limitation was due to fast growth responses of a few N- limited species such as the diatom Chaetoceros wighamii (Brightwell) and the mixotrophic chrysophyte Uroglena sp. Another dominating diatom, Skeletonema costatum (Greville) Cleve was most clearly P-limited. The N:P ratio had the strongest effect on Uroglena sp., which grew exponentially in the enrichments with a high N:P ratio. This can be explained by the ability of the species to feed on P-rich bacteria, which gives it a competitive advantage in P-limited conditions. The species-specific differences in the responses to the nutrient enrichments can generally be explained by differences in the species physiology and they were consistent with the theory of resource competition.
While searching for prey, Molossus molossus broadcasts narrow-band calls of 11.42 ms organized in pairs of pulses that alternate in frequency. The first signal of the pair is at 34.5 kHz, the second at 39.6 kHz. Pairs of calls with changing frequencies were only emitted when the interpulse intervals were below 200 ms. Maximum duty cycles during search phase are close to 20%. Frequency alternation of search calls is interpreted as a mechanism for increasing duty cycle and thus the temporal continuity of scanning, as well as increasing the detection range. A neurophysiological correlate for the processing of search calls was found in the inferior colliculus. 64% of neurons respond to frequencies in the 30- to 40-kHz range and only in this frequency range were closed tuning curves found for levels below 40 dB SPL. In addition, 15% of the neurons have double-tuned frequency-threshold curves with best thresholds at 34 and 39 kHz. Differing from observations in other bats, approach calls of M. molossus are longer and of higher frequencies than search calls. Close to the roost, the call frequency is increased to 45.049.8 kHz and, in addition, extremely broadband signals are emitted. This demonstrates high plasticity of call design
Nonmuscle myosin-II is a motor protein that drives cell movement and changes in cell shape during tissue and organ development. This study has determined he dynamic changes in myosin-II distribution during Drosophila compound eye morphogenesis. In photoreceptor neurons, myosin-II is undetectable at the apical domain throughout the first half of pupal life, at which time this membrane domain is involuted into the epithelium and progresses toward the retinal floor. Myosin-II is deployed at the apical surface at about 60% of pupal development, once the developing rhabdomeres reach the retinal floor. Subsequently, myosin-II becomes restricted to two stripes at the sides of the developing rhabdomere, adopting its final position within the visual cells R1-6; here, myosin-II is associated with a set of actin filaments that extend alongside the rhabdomeres. At the midpupal stage, myosin-II is also incorporated into stress-fiber-like arrays within the basal endfeet of the pigment cells that then change their shape. This spatiotemporal pattern of myosin- II localization and the morphological defects observed in the eyes of a myosin-II mutant suggest that the myosin-II/F- actin system is involved in the alignment of the rhabdomeres within the retina and in the flattening of the retinal floor. The observation that the myosin-II/F-actin arrays are incomplete or disorganized in R7/R8 and in rhodopsin-1-null R1-6 suggests further that the establishment and stability of this cytoskeletal system depend on rhodopsin-1 expression. (C) 2004 Elsevier Inc. All rights reserved
Ecologists increasingly use spatial statistics to study vegetation patterns. Mostly, however, these techniques are applied in a purely descriptive fashion without a priori statements on the pattern characteristics expected. We formulated such a priori predictions in a study of spatial pattern in a semi-arid Karoo shrubland, South Africa. Both seed dispersal and root competition have been discussed as processes shaping the spatial structure of this community. If either of the two processes dominates pattern formation, patterns within and between shrub functional groups are expected to show distinct deviations from null models. We predicted the type and scale of these deviations and compared predicted to observed pattern characteristics. As predicted by the seed dispersal hypothesis, small-scale co-occurrence within and between groups of colonisers and successors was increased as compared to complete spatially random arrangement of shrubs. The root competition predictions, however, were not met as shrubs of similar rooting depth co- occurred more frequently than expected under random shrub arrangement. Since the distribution of rooting groups to the given shrub locations also failed to match the root competition predictions, there was little evidence for dominance of root competition in pattern formation. Although other processes may contribute to small-scale plant co-occurrence, the sufficient and most parsimonious explanation for the observed pattern is that its formation was dominated by seed dispersal. To characterise point patterns we applied both cumulative (uni- and bivariate K-function) and local (pair- and mark-correlation function) techniques. Based on our results we recommend that future studies of vegetation patterns include local characteristics as they independently describe a pattern at different scales and can be easily related to processes changing with interplant distance in a predictable fashion.
The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic a helices of the protein. Less is known about the significance of the loop domains connecting the a helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088- 14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses. we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix
Serotonin and histamine produce different spatiotemporal Ca2+ signals in blowfly salivary glands
(2004)
Standing crop and species composition in semiarid grassland are linked to long-term patterns of water availability, but grasslands are characterized by large single-season variability in rainfall. We tested whether a single season of altered water availability influenced the proportions of grasses and shrubs in a semiarid grassland near the northern edge of the North American Great Plains. We studied stands of the clonal shrub snowberry (Symphoricarpos occidentalis) and adjacent grassland dominated by the native grasses Stipa spartea and Bouteloua gracilis. Rain was excluded and water supplied in amounts corresponding to years of low, medium, and high rainfall, producing a 2 - 4-fold range in monthly precipitation among water supply treatments. There were ten replicate plots of each water treatment in both snowberry stands and grassland. Grass standing crop increased significantly with water availability in grassland but not inside snowberry stands. Total standing crop and shrub stem density increased significantly with water supply, averaged across both communities. In contrast, water had no effect on shrub standing crop or light penetration. In summary, our finding that water has significant effects on a subset of components of grassland vegetation is consistent with long-term, correlational studies, but we also found that a single season of altered water supply had no effect on other important aspects of the ecosystem.
Shrub encroachment, i.e. the increase in woody plant cover, is a major concern for livestock farming in southern Kalahari savannas. We developed a grid-based computer model simulating the population dynamics of Grewia flava, a common, fleshy-fruited encroaching shrub. In the absence of large herbivores, seeds of Grewia are largely deposited in the sub-canopy of Acacia erioloba. Cattle negate this dispersal limitation by browsing on the foliage of Grewia and dispersing seeds into the grassland matrix. In this study we first show that model predictions of Grewia cover dynamics are realistic by comparing model output with shrub cover estimates obtained from a time series of aerial photographs. Subsequently, we apply a realistic range of intensity of cattle-induced seed dispersal combined with potential precipitation and fire scenarios. Based on the simulation results we suggest that cattle may facilitate shrub encroachment of Grewia. The results show that the severity of shrub encroachment is governed by the intensity of seed dispersal. For a high seed dispersal intensity without fire (equivalent to a high stocking rate) the model predicts 56% shrub cover and 85% cell cover after 100 yr. With fire both recruitment and shrub cover are reduced, which may, under moderate intensities, prevent shrub encroachment. Climate change scenarios with two-fold higher frequencies of drought and wet years intensified shrub encroachment rates, although long-term mean of precipitation remained constant. As a management recommendation we suggest that shrub encroachment on rangelands may be counteracted by frequent fires and controlling cattle movements to areas with a high proportion of fruiting Grewia shrubs
Pectins are major components of primary plant cell walls and the seed mucilage of Arabidopsis. Despite progress in the structural elucidation of pectins, only very few enzymes participating in or regulating their synthesis have been identified. A first candidate gene involved-in the synthesis of pectinaceous rhamnogalacturonan I is RHM2, a putative plant ortholog to NDP-rhamnose biosynthetic enzymes in bacteria. Expression studies with a promoter beta-glucuronidase construct and reverse transcription PCR data show that RHM2 is expressed ubiquitously. Rhm2 T-DNA insertion mutant lines were identified using a reverse genetics approach. Analysis of the rhm2 seeds by various staining methods and chemical analysis of the mucilage revealed a strong reduction of rhamnogalacturonan I in the mucilage and a decrease of its molecular weight. In addition, scanning electron microscopy of the seed surface indicated a distorted testa morphology, illustrating not only a structural but also a developmental role for RGI or rhamnose metabolism in proper testa formation
Rapid decay of diversity-productivity relationships after invasion of experimental plant communities
(2004)
The small fox tapeworm (Echinococcus multilocularis) shows a heterogeneous spatial distribution in the intermediate host (Microtus arvalis). To identify the ecological processes responsible for this heterogeneity, we developed a spatially explicit simulation model. The model combines individual-based (foxes, Vulpes vulpes) and grid- based (voles) techniques to simulate the infections in both intermediate and definite host. If host populations are homogeneously mixed, the model reproduces field data for parasite prevalence only for a limited number of parameter combinations. As ecological parameters inevitably vary to a certain degree, we discarded the homogeneous mixing model as insufficient to gain insight into the ecology of the fox tapeworm cycle. We analysed five different model scenarios, each focussing on an ecological process that might be responsible for the heterogeneous spatial distribution of E multilocularis in the intermediate host. Field studies revealed that the prevalence ratio between intermediate and definite host remains stable over a wide range of ecological conditions. Thus, by varying the parameters in simulation experiments, we used the robustness of the agreement between field data and model output as quality criterion for the five scenarios. Only one of the five scenarios was found to reproduce the prevalence ratio over a sufficient range of parameter combinations. In the accentuated scenario most tapeworm eggs die due to bad environmental conditions before they cause infections in the intermediate host. This scenario is supported by the known sensitivity of tapeworm eggs to high temperatures and dry conditions. The identified process is likely to lead to a heterogeneous availability of infective eggs and thus to a clumped distribution of infected intermediate hosts. In conclusion, areas with humid conditions and low temperatures must be pointed out as high risk areas for human exposure to E. multilocularis eggs as well. (C) 2004 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved
Predicting evolution of floral traits associated with mating system in a natural plant population
(2004)
Evolution of floral traits requires that they are heritable, that they affect fitness, and that they are not constrained by genetic correlations. These prerequisites have only rarely been examined in natural populations. For Mimulus guttatus, we found by using the Riska-method that corolla width, anther length, ovary length and number of red dots on the corolla were heritable in a natural population. Seed production (maternal fitness) was directly positively affected by corolla width and anther size, and indirectly so by ovary length and number of red dots on the corolla. The siring success (paternal fitness), as estimated from allozyme data, was directly negatively affected by anther-stigma separation, and indirectly so by the corolla length-width ratio. Genetic correlations, estimated with the Lynch-method, were positive between floral size measures. We predict that larger flowers with larger reproductive organs, which generally favour outcrossing, will evolve in this natural population of M. guttatus
A comprehensive study gives experimental evidence that a complex made from pyrrole and beta-naphthalenesulfonic acid in a molar composition of 3:1 acts as morphological precursor in the subsequent oxidative polymerization of pyrrole initiated with ammonium peroxodisulfate. The precursor complex itself is unable to polymerize but its outer parallelepipedal shape with a high aspect ratio is templated in the inner surface of the formed conducting polypyrrole tubes
Amyloid protofibril formation of phosphoglycerate kinase (PGK) and Syrian hamster prion protein (SHaPrP(90- 232)) were investigated by static and dynamic light scattering, size exclusion chromatography and electron microscopy. Changes in secondary structure were monitored by Fourier transform infrared spectroscopy and by circular dichroism. Protofibril formation of the two proteins is found to be a two-stage process. At the beginning, an ensemble of critical oligomers is built lip. These critical oligomeric states possess a predominant beta-sheet structure and do not interact considerably with monomers. Initial oligomerization and transition to beta-sheet structure are coupled events differing in their details for both proteins. Intermediate oligomeric states (dimers, trimers, etc.) are populated in case of PGK, whereas SHaPrP(90-232) behaves according to oil apparent two-state reaction between monomers and octamers rich in beta- structure with a reaction order varying between 2 and 4. All oligomers coalesce to PGK protofibrils in the second stage, while SHaPrP(90-232) protofibrils are only formed by a subpopulation. The rates of both growth stages can be tuned in case of PGK by different salts preserving the underlying generalized diffusion-collision mechanism. The different kinetics of the early misfolding and oligomerization events of the two proteins argue against a common mechanism of protofibril formation. A classification scheme for misassembly, mechanisms of proteins based on energy landscapes is presented. It includes scenarios of downhill polymerization to which protofibril formation of PGK and SHaPrP(90-232) belong
The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied P-32 orthophosphate into starch. Illuminated cells incorporated P-32 into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with P-32/P-31 orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch
Partikelverstärkte Oberflächenplasmonresonanz für die Quantifizierung von Matrix Metalloproteinase-2
(2004)
Offenhaltung durch Feuer
(2004)
Negative effects of P-buffering and pH on photosynthetic activity of planktonic desmid species
(2004)
The photosynthetic activities of three planktonic desmid species (Staurastrum brachiatum, Staurodesmus cuspidatus var. curvatus, and Staurastrum chaetoceras) were compared after adaptation to medium enriched with either a 20 mM Na+- phosphate (P) or HEPES buffer. Incubations up to 2 d were carried out at pH 6 or 8 under normal air or air enriched with 5 % CO2. Gross maximum photosynthetic rate (Pmax) and growth rate were decreased in both S. brachiatum and Std. cuspidatus at higher pH when using the HEPES buffer and this effect was independent of CO2 concentration, indicating that pH had an inhibitory effect on photosynthesis and growth in these species. The P-buffer at pH 8 caused a large decrease in Pmax and quantum yield for charge separation in photosystem 2 (PS2), compared to HEPES-buffered algae. This effect was very large in both S. brachiatum and Std. cuspidatus, two species characteristic of soft water lakes, but also significant in S. chaetoceras, a species dominant in eutrophic, hard water lakes. The decreased Pmax in P- buffer could not be related to a significant increase in cellular P content known to be responsible for inhibition in isolated chloroplasts. Experiments at pH 6 and 8 showed that two conditions, high pH and high Na+ concentration, both contributed to the decreased Pmax and quantum yield in the desmids. Effects of a P-buffer were less pronounced by using K+-P buffer. The use of P-buffer at pH 8 possibly resulted in high irradiance stress in all species, indicated by damage in the PS2 core complex. In the soft water species pH 8 resulted in increased non-photochemical quenching together with a high de-epoxidation state of the xanthophyll cycle pigments.
In northeastern German pine forests we studied re-colonization patterns of experimental gaps by four dominant bryophyte species (Dicranum scoparium, Hypnum jutlandicum, Pleurozium schreberi and Scleropodium purum) over three years. Both vegetation and litter layer were removed on 1 m(2) plots within +/- pure colonies of the experimental species, while the humus layer was left intact. All plots were vegetatively re-colonized by the species which was dominant before gap creation. Three mechanisms of re-colonization occurred and interacted: (1) advance of surrounding shoots from the edge into the gaps by clonal growth, (2) dispersal of detached single shoots as well as larger clumps of multiple shoots into the plots, resulting in new colonies by continuing growth, and (3) regeneration from a soil diaspore bank consisting of seemingly dead stem fragments in the humus layer of the gaps. Scleropodium purum, which occurs at locations with good water and nutrient supply, displayed the most rapid growth. Here, some plots were completely recovered after three years. Despite lower rates of advance from the edge, colonization of Hypnum jutlandicum was faster than and of Dicranum scoparium as fast than that of Pleurozium schreberi because of a larger diaspore bank. Thus, each bryophyte species was characterized by a different habitat occupation strategy. The different clonal colonization strategies account for the high competitive capacity and regeneration potential of the investigated bryophyte species in pine forests despite of the lack of generative reproduction. Experimental disturbance resulted in a temporary increase of bryophyte diversity, because short-lived Colonists with a low competitive capacity colonized the gaps, before they will be overgrown by the dominant Perennials
Molinio-Arrhenatheretea = Kulturgrasland und verwandte Vegetationstypen. Teil 2: Molinietalia
(2004)
Environmental stresses such as drought, high salt and low temperature affect plant growth and decrease crop productivity extremely. It is important to improve stress tolerance of the crop plant to increase crop yield under stress conditions. The Arabidopsis thaliana salt tolerance 1 gene (AtSTO1) was originally identified by Lippuner et al., (1996). In this study around 27 members of STO-like proteins were identified in Arabidopsis thaliana, rice and other plant species. The STO proteins have two consensus motifs (CCADEAAL and FCV(L)EDRA). The STO family members can be regarded as a distinct class of C2C2 proteins considering their low sequence similarity to other GATA like proteins and poor conservation in the C-terminus. AtSTO1 was found to be induced by salt, cold and drought in leaves and roots of 4-week-old Arabidopsis thaliana wild-type plants. The expression of AtSTO1 under salt and cold stress was more pronounced in roots than in leaves. The data provided here revealed that the AtSTO1 protein is localized in the nucleus. The observation that AtSTO1 localizes in the nucleus is consistent with its proposed function as a transcription factor. AtSTO1-dependent phenotypes were observed when plant were grown at 50 mM NaCl on agar plates. Leaves of AtSTO1 overexpression lines were bigger with dark green coloration, whereas stunted growth and yellowish leaves were observed in wild-type and RNAi plants. Also, the AtSTO1 overexpression plants when exposed to long-term cold stress had a red leaf coloration which was much stronger than in wild-type and RNAi lines. Growth of AtSTO1 overexpression lines in long term under salt and cold stress was always associated with long roots which was more pronounced than in wild-type and RNAi lines. Proline accumulation increased more strongly in leaves and roots of AtSTO1 overexpression lines than in tissues of wild-type and RNAi lines when treated with 200 mM NaCl, exposed to cold stress or when watering was prevented for one day or two weeks. Also, soluble sugar content increased to higher levels under salt, cold and drought stress in AtSTO1 overexpression lines when compared to wild-type and RNAi lines. The increase in soluble sugar content was detected in AtSTO1 overexpression lines after long-term (2 weeks) growth of plants under these stresses. Anthocyanins accumulated in leaves of AtSTO1 overexpression lines when exposed to long term salt stress (200 mM NaCl for 2 weeks) or to 4°C for 6 and 8 weeks. Also, anthocyanin content was increased in flowers of AtSTO1 overexpression plants kept at 4°C for 8 weeks. Taken together these data indicate that overexpression of AtSTO1 enhances abiotic stress toleranc via a more pronounced accumulation of compatible solutes under stress.
Molecular and physiological analyses of diacylglycerol kinases from arabidopsis thaliana (L.) Heynh.
(2004)
Evolutionary relationships of different populations of the threatened malagasy lemur Lepilemur septentrimialis were assessed by sequence analysis of mitochondrial DNA (D-loop region and partial Cyt b gene). One hundred and fifty nine samples were collected from five main different localities in the northern part of Madagascar. We applied the phylogenetic species concept based on fixed diagnostic differences to determine the status of different geographical populations. No nucleotide site diagnoses Ankarana from Andrafiamena or Analamera. However, numerous fixed differences separate Sahafary from all other populations. These results were corroborated by phylogenetic trees. As previous cytogenetic studies, our molecular data suggest that two cryptic species of Lepilemur occur in the extreme north of Madagascar. This speciation is probably caused by chromosomal rearrangements in at least one of the evolutionary lineages. Our study comprises another striking example of how molecular genetic assay can detect phylogenetic discontinuities that are not reflected in traditional morphologically based taxonomies. Our study indicates that the Sahafary population is a hitherto undescribed endangered endemic species which urgently needs conservation efforts. (C) 2003 Elsevier Inc. All rights reserved
In ecology much attention has been paid towards seed dispersal of fleshy-fruited plants, however, knowledge is lacking about the Iona-term demographic consequences of variation in dispersal distance and fruit removal rate, particularly given the natural variability of the environment the organism lives in. In this study we used a spatially explicit, two-level stochastic computer model to simulate population dynamics of a fleshy-fruited shrub living in the sub-canopy of solitary savanna trees. On the landscape level we implemented three realistic scenarios of savanna landscape dynamics for a period of 500 years with equal inter-annual mean of environmental variables. The first scenario is representative of a relatively constant environment with normal variability in precipitation, constant tree density and random tree recruitment pattern. The second and third scenarios represent positive auto-correlated, cyclic patterns with alternating phases of tree cover increase and decrease corresponding with favorable and unfavorable rain phases. Our simulation experiments show that when fruit removal rate is extremely low, population persistence is enhanced under relatively constant rain conditions, while alternating rain phases of the cyclic scenarios lead to a significant population decrease. This result confirms previous findings that periodically fluctuating environments may increase local extinction risk. However, when dispersal distance is a limiting factor (whilst removal rate was sufficiently high), tree clumps typically forming in wet phases of both cyclic scenarios compensated for the negative effect of low dispersal distances, while the constant scenario with random tree pattern and larger inter-tree distances resulted in a significant population decline. (C) 2003 Elsevier B.V. All rights reserved
The overall objective of the study is an elaboration of quantitative methods for national conservation planning, coincident with the international approach ('hotspots' approach). This objective requires a solution of following problems: 1) How to estimate large scale vegetation diversity from abiotic factors only? 2) How to adopt 'global hotspots' approach for bordering of national biodiversity hotspots? 3) How to set conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots? 4) How to design large scale national conservation plan reflecting hierarchical nature of biodiversity? The case study for national conservation planning is Russia. Conclusions: · Large scale vegetation diversity can be predicted to a major extent by climatically determined latent heat for evaporation and geometrical structure of landscape, described as an altitudinal difference. The climate based model reproduces observed species number of vascular plant for different areas of the world with an average error 15% · National biodiversity hotspots can be mapped from biotic or abiotic data using corrected for a country the quantitative criteria for plant endemism and land use from the 'global hotspots' approach · Quantitative conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots can be set using national data for Red Data book species · Large scale national conservation plan reflecting hierarchical nature of biodiversity can be designed by combination of abiotic method at national scale (identification of large scale hotspots) and biotic method at regional scale (analysis of species data from Red Data book)
To unravel the postglacial colonization history and the current intercolony dispersal in the common eider, Somateria mollissima, we analysed genetic variation at a part of the mitochondrial control region and five unlinked autosomal microsatellite loci in 175 eiders from 11 breeding colonies, covering the entire European distribution range of this species. As a result of extreme female philopatry, mitochondrial DNA differentiation is substantial both among local colonies and among distant geographical regions. Our study further corroborates the previous hypothesis of a single Pleistocene refugium for European eiders. A nested clade analysis on mitochondrial haplotypes suggests that (i) the Baltic Sea eider population is genetically closest to a presumably ancestral population and that (ii) the postglacial recolonization progressed in a stepwise fashion via the North Sea region and the Faroe Islands to Iceland. Current long-distance dispersal is limited. Differentiation among colonies is much less pronounced at microsatellite loci. The geographical pattern of this nuclear genetic variation is to a large extent explained by isolation by distance. As female dispersal is very limited, the geographical pattern of nuclear variation is probably explained by male-mediated gene flow among breeding colonies. Our study provides genetic evidence for the assumed prominent postglacial colonization route shaping the present terrestrial fauna of the North Atlantic islands Iceland and the Faroes. It suggests that this colonization had been a stepwise process originating in continental Europe. It is the first molecular study on eider duck populations covering their entire European distribution range
The possibly distinct Carpathian red deer was compared genetically to other European populations. We screened 120 red deer specimens from Serbia, the Romanian lowland and the Romanian Carpathians for genetic variability using 582 bp of the mitochondrial control region and nine polymorphic nuclear microsatellite loci. The study aimed at a population genetic characterization of the Carpathian red deer, which are often treated as a distinct subspecies (Cervus elaphus montanus). The genetic integrity of the Carpathian populations was confirmed through the haplotype distribution, private alleles and genetic distances. The Carpathian red deer are thus identified as one of the few remaining natural populations of this species, deserving special attention among game and conservation biologists. The history of the populations studied, in particular the introduction of Carpathian red deer into Romanian lowland areas in the 20th century, was reflected by the genetic data
There is concern about the lack of recruitment of Acacia trees in the Negev desert of Israel. We have developed three models to estimate the frequency of recruitment necessary for long-term population survival (i.e. positive average population growth for 1,000 years and <10% probability of extinction). Two models assume purely episodic recruitment based on the general notion that recruitment in and environments is highly episodic. They differ in that the deterministic model investigates average dynamics while the stochastic model does not. Studies indicating that recruitment episodes in and environments have been overemphasized motivated the development of the third model. This semi-stochastic model simulates a mixture of continuous and episodic recruitment. Model analysis was done analytically for the deterministic model and via running model simulations for the stochastic and semi-stochastic models. The deterministic and stochastic models predict that, on average, 2.2 and 3.7 recruitment events per century, respectively, are necessary to sustain the population. According to the semi-stochastic model, 1.6 large recruitment events per century and an annual probability of 50% that a small recruitment event occurs are needed. A consequence of purely episodic recruitment is that all recruitment episodes produce extremely large numbers of recruits (i.e. at odds with field observations), an evaluation that holds even when considering that rare events must be large. Thus, the semi- stochastic model appears to be the most realistic model. Comparing the prediction of the semi-stochastic model to field observations in the Negev desert shows that the absence of observations of extremely large recruitment events is no reason for concern. However, the almost complete absence of small recruitment events is a serious reason for concern. The lack of recruitment may be due to decreased densities of large mammalian herbivores and might be further exacerbated by possible changes in climate, both in terms of average precipitation and the temporal distribution of rain
A seed sowing experiment was conducted in a mixed secondary woodland on acidic soils in NE Germany with Melampyrum pratense, an annual ant-dispersed forest herb which lacks a natural population in the study area, but is abundant in similar habitats. Each set of 300 seeds was sown within one square metre at three sites in 1997, and the development of the populations was recorded from 1998 onward. Additionally, seed fall patterns were studied in a natural population by means of adhesive cardboard. All trials resulted in the recruitment of populations, which survived and increased in both individual number and area, up to the year 2001. Thus, local distribution of Melampyrum pratense is dispersallimited. Total individual number increased from 105 to 3,390, and total population area from 2.07 to 109.04 m². Migration occurred in all directions. Mean migration rate was 0.91 m per year, and the highest migration rate was 6.48 m. No individual was recorded beyond 7.63 m from the centres of the sawn squares after three years, suggesting exclusive short-distance dispersal. As primary dispersal enables only distances of up to 0.25 m, ants are presumed to be the main dispersal vectors. Despite differences in individual number and colonization patterns, migration rates did not differ significantly between the populations, but were significantly higher in 2001 due to an increased population size. Colonization patterns were characterized by a rapid, negative exponential decrease of population density with increasing distance from the sown plot, suggesting a colonization by establishment of more or less isolated outposts of individuals and a subsequent gradual infill of the gaps between. My results resemble myrmecochorous dispersal distances in temperate woodlands, and migration rates and patterns across ecotones from ancient to recent deciduous forests. They may function as a colonization model of Melampyrum pratense after accidental long-distance dispersal.
1. Lakes formed in mining pits often contain high concentrations of dissolved ferric iron and sulphate (e.g. 2 and 16 mmol L)1, respectively) and the pH is buffered between 2.5 and 3.5. Efforts to neutralise their water are based on the stimulation of lake internal, bacterial iron- and sulphate reduction. Electron donors may be supplied by organic carbon compounds or indirectly by enhancement of primary production. Here, we investigated the function of mixotrophic algae, which can potentially supplement or deplete the organic carbon pool, in the carbon metabolism and alkalinity budget of an acidic mining lake. 2. Two weeks after organic substrates had been added in a large in situ mesocosm of 30 m diameter, a bloom of Chlamydomonas occurred, reaching a biovolume of 80 mm3 L)1. Growth experiments using filtered lake water showed that the alga reduced the overall dissolved organic carbon (DOC) concentration despite significant photosynthetic activity. However, when Chlamydomonas were grown together with natural bacterioplankton, net DOC consumption did not increase. 3. Uptake experiments using [14C]-glucose indicated that bacteria dominated glucose uptake and remineralisation. Therefore, the DOC leached in the water column was processed mainly by planktonic bacteria. Leached DOC must be regarded as loss, not transferred by larger organisms to the sediment, where reduction processes take place. 4. From phytoplankton biomass and production 2 years after fertilisation we estimated that pelagic photosynthesis does not supply an electron donor capacity capable of reducing more than 2% of actual stock of acidity per year. We estimated that only the benthic primary production was in a range to compensate for ongoing inputs of iron and sulphate.
Local adaption of the clonal plant Ranunculus reptans to flooding along a small-scale gradient.
(2004)
Contents: Introduction Experimental Techniques: The LIF demonstrator unit - The LIF demonstrator unit - The mobile LIF spectrometer OPTIMOS - Investigated petroleum products and soil samples Results and Discussion: Photophysical properties of the petroleum products LIF spectroscopic investigations of oil-spiked samples LIF spectroscopic investigations of real-world soils Conclusions
Investigating of homophilic interactions of the tight junction proteins occludin and claudin-5
(2004)
The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse.
The past decades are characterized by various efforts to provide complete sequence information of genomes regarding various organisms. The availability of full genome data triggered the development of multiplex high-throughput assays allowing simultaneous measurement of transcripts, proteins and metabolites. With genome information and profiling technologies now in hand a highly parallel experimental biology is offering opportunities to explore and discover novel principles governing biological systems. Understanding biological complexity through modelling cellular systems represents the driving force which today allows shifting from a component-centric focus to integrative and systems level investigations. The emerging field of systems biology integrates discovery and hypothesis-driven science to provide comprehensive knowledge via computational models of biological systems. Within the context of evolving systems biology, investigations were made in large-scale computational analyses on transcript co-response data through selected prokaryotic and plant model organisms. CSB.DB - a comprehensive systems-biology database - (http://csbdb.mpimp-golm.mpg.de/) was initiated to provide public and open access to the results of biostatistical analyses in conjunction with additional biological knowledge. The database tool CSB.DB enables potential users to infer hypothesis about functional interrelation of genes of interest and may serve as future basis for more sophisticated means of elucidating gene function. The co-response concept and the CSB.DB database tool were successfully applied to predict operons in Escherichia coli by using the chromosomal distance and transcriptional co-responses. Moreover, examples were shown which indicate that transcriptional co-response analysis allows identification of differential promoter activities under different experimental conditions. The co-response concept was successfully transferred to complex organisms with the focus on the eukaryotic plant model organism Arabidopsis thaliana. The investigations made enabled the discovery of novel genes regarding particular physiological processes and beyond, allowed annotation of gene functions which cannot be accessed by sequence homology. GMD - the Golm Metabolome Database - was initiated and implemented in CSB.DB to integrated metabolite information and metabolite profiles. This novel module will allow addressing complex biological questions towards transcriptional interrelation and extent the recent systems level quest towards phenotyping.
Die immunologische Kontrazeption mittels Zona pellucida (ZP) Proteinen gilt als vielversprechender Ansatz für die Reproduktionskontrolle verwilderter Haus- und Wildtierbestände. Da die Applikation von nativer ZP mit Nebenwirkungen verbunden ist, wird die Verwendung einzelner ZP Peptide als Bestandteil kontrazeptiver Vakzine als besonders aussichtsreich erachtet. Das Prinzip dieser nebenwirkungsfreien ZP Immunisierung ist die gezielte Trennung der Entzündungsreaktionen auslösenden T-Zell-Epitope der ZP von den kontrazeptiv wirkenden B-Zell-Epitopen. Niedermolekulare synthetische oder rekombinante Peptide allein sind gering immunogen und können somit keine ausreichende Immunantwort induzieren. Die Verwendung von Peptiden für die immunologische Kontrazeption erfordert daher ein „Vakzin-Design“, d. h. die gezielte Kombination der Peptide mit immunstimulierenden Substanzen (Liposomen, Carrierproteinen, Adjuvantien). Zielstellung der vorliegenden Arbeit war die Untersuchung des Potentials synthetischer Peptide für die Immunokontrazeption von verwilderten Hauskatzen (Felis catus). Dazu wurden zunächst relevante B-Zell-Epitope des felinen Zona pellucida Proteins, ZPB2, identifiziert und synthetisiert. Zwei der synthetischen Peptide (P3, P6) wurden zur Herstellung von Antikörpern an BSA konjugiert und zusammen mit Freundschem Adjuvans in Ratten verimpft. Die kontrazeptive Relevanz beider Peptide sowie der Ratten Anti-Peptid Antiseren wurde im in vitro Befruchtungssystem der Hauskatze geprüft. Zur Untersuchung der Immunogenität der Peptide in der Zielspezies Hauskatze erfolgte die Entwicklung von Vakzin-Prototypen für die einmalige Applikation. Neben der Eruierung der Stärke und Dauer der Immunantwort wurde durch Verpaarung der Tiere auch das kontrazeptive Potential in vivo abgeschätzt.
Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.
For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.
Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase beta-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as beta- mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N- terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a K-a of 1 x 10(7) M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55 Angstrom and 1.06 Angstrom resolution, respectively. In the crystal, CsCBM27-1 shows the typical beta-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs. (C) 2004 Elsevier Ltd. All rights reserved
Heterosis has been widely used in agriculture to increase yield and to broaden adaptability of hybrid varieties and is applied to an increasing number of crop species. We performed a systematic survey of the extent and degree of heterosis for dry biomass in 63 Arabidopsis accessions crossed to three reference lines (Col-0, C24, and Nd). We detected a high heritability (69%) for biomass production in Arabidopsis. Among the 169 crosses analyzed, 29 exhibited significant mid-parent-heterosis for shoot biomass. Furthermore, we analyzed two divergent accessions, C24 and Col-0, the F-1 hybrids of which were shown to exhibit hybrid vigor, in more detail. In the combination Col-0/C24, heterosis for biomass was enhanced at higher light intensities; we found 51% to 66% mid-parent-heterosis at low and intermediate light intensities (60 and 120 mumol m(-2) s(-1)), and 161% at high light intensity (240 mumol m(-2) s(-1)). While at the low and intermediate light intensities relative growth rates of the hybrids were higher only in the early developmental phase (0-15 d after sowing [DAS]), at high light intensity the hybrids showed increased relative growth rates over the entire vegetative phase (until 25 DAS). An important finding was the early onset of heterosis for biomass; in the cross Col-0/C24, differences between parental and hybrid lines in leaf size and dry shoot mass could be detected as early as 10 DAS. The widespread occurrence of heterosis in the model plant Arabidopsis opens the possibility to investigate the genetic basis of this phenomenon using the tools of genetical genomics