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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
Mountain gazelles (Gazella gazella) rank among the most critically endangered mammals on the Arabian Peninsula. Past conservation efforts have been plagued by confusion about the phylogenetic relationship among various 'phenotypically discernable' populations, and even the question of species boundaries was far from being certain. This lack of knowledge has had a direct impact on conservation measures, especially ex situ breeding programmes, hampering the assignment of captive stocks to potential conservation units. Here, we provide a phylogenetic framework, based on the analysis of mtDNA sequences (360 bp cytochrome b and 213 bp Control Region) of 126 individuals collected from the wild throughout the Arabian Peninsula and from captive stocks. Our analyses revealed two reciprocally monophyletic genetic lineages within the presumed species Gazella gazella: one 'northern clade' on the Golan Heights (Israel/Syrian border) and one genetically diverse larger clade from the rest of the Arabian Peninsula including the Arava Valley (Negev, Israel). Applying the Strict Phylogenetic Species Concept (sensu Mishler & Theriot, 2000) allows assigning species status to these two major clades.
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes
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
Reaction centers (RCs) of purple bacteria are uniquely suited objects to study the mechanisms of the photosynthetic conversion of light energy into chemical energy. A recently introduced method of higher order derivative spectroscopy [I.K. Mikhailyuk, H. Lokstein, A.P. Razjivin, A method of spectral subband decomposition by simultaneous fitting the initial spectrum and a set of its derivatives, J. Biochem. Biophys. Methods 63 (2005) 10-23] was used to analyze the NIR absorption spectra of RC preparations from Rhodobacter (R.) sphaeroides strain 2R and Blastochloris (B.) viridis strain KH, containing bacteriochlorophyll (BChl) a and b, respectively. Q(y) bands of individual RC porphyrin components (BChls and bacteriopheophytins, BPheo) were identified. The results indicate that the upper exciton level Py+ of the photo-active BChl dimer in RCs of R. sphaeroides has an absorption maximum of 810nm. The blue shift of a complex integral band at approximately 800nm upon oxidation of the RC is caused primarily by bleaching of Py+, rather than by an electrochromic shift of the absorption band(s) of the monomeric BChls. Likewise, the disappearance of a band peaking at 842 nm upon oxidation of RCs from B. viridis indicates that this band has to be assigned to Py+, A blue shift of an absorption band at approximately 830nm upon oxidation of RCs of B. viridis is also essentially caused by the disappearance of Py+, rather than by an electrochromic shift of the absorption bands of monomeric BChls. Absorption maxima of the monomeric BCHls, B-B and B-A are at 802 and 797nm, respectively, in RCs of R. sphaeroides at room temperature. BPheo co-factors H-B and HA peak at 748 and 758 nm, respectively, at room temperature. For B. viridis RCs the spectral positions of HB and HA were found to be 796 and 816nm, respectively, at room temperature.
We examined factors predicting female densities within the common home ranges of related females ("clans") in bushbuck Tragelaphus scriptus Pallas, 1776. In this species, each female forms an individual home range, but the home ranges of matrilineal clan members strongly overlap. We found female densities to increase in areas with high canopy cover. Moreover, individual home range sizes tended to decrease with increasing cover. Food plant availability and intruder pressure by two heterospecific competitors did not significantly affect female densities. Apparently, canopy cover is the major limited resource in this species. A possible explanation is that both adult bushbuck and - even more markedly - fawns hide from predators in dense vegetation, in particular in thicket clumps and coalescences. The study shows an effect of habitat properties (eg sufficient canopy cover) on a within-population level in bushbuck, where female densities differ even among proximate clan areas
Metabolomic networks in plants : transitions from pattern recognition to biological interpretation
(2006)
Nowadays techniques for non-targeted metabolite profiling allow for the generation of huge amounts of relevant data essential for the construction of dynamic metabolomic networks. Thus, metabolomics, besides transcriptomics or proteomics, provides a major tool for the characterization of postgenomic processes. In this work, we introduce comparative correlation analysis as a complementary approach to characterize the physiological states of various organs of diverse plant species with focus on specific participation of metabolites in different reaction networks. The correlations observed are induced by diminutive fluctuations in environmental conditions, which propagate through the system and induce specific patterns depending on the genomic background. In order to examine this hypothesis, numeric examples of such fluctuations are computed and compared with experimentally obtained metabolite data.
Atlantic mollies (Poecilia mexicana) inhabit a variety of surface habitats, but they also occur in a sulfur cave in southern Mexico. We examined male mate choice relative to female body size in the cave population and in the most closely related surface-dwelling population from a nearby river. Males from both populations were either light- or dark-reared and could choose between two differently sized females either on the basis of visual cues in light or on the basis of solely nonvisual cues in darkness. Sexual preferences were estimated from the degree of association. Cave molly males always showed a preference for the larger female, both in light and in darkness. Among the surface males, only light-reared males showed a preference in the visual cues test, but not in darkness. In a control experiment, we demonstrated that male association preferences directly translate into actual mating preferences. Apparently, using visual cues for mate choice is the ancestral state in this system, and using nonvisual cues has evolved as a novel trait in the cave population. We discuss the evolution of nonvisual male mate choice in the context of changed environmental conditions, namely the absence of light, hypoxia, and toxic hydrogen sulfide in the cave
The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAc alpha 1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing