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Males often face strong mating competition by neighboring males in their social environment. A recent study by Plath et al. (Anim Behav 75:21-29, 2008a) has demonstrated that the visual presence of a male competitor (i.e., an audience male) affects the expression of male mating preferences in a poeciliid fish (Poecilia mexicana) with a weaker expression of mating preferences when an audience male observed the focal male. This may be a tactic to reduce sperm competition, since surrounding males likely share intrinsic preferences for female traits or copy mate choice decisions. Here, we examined the hypothesis that a same-sex audience would affect female mate preferences less than male mating preferences. Our hypothesis was based on the assumptions that (1) competition for mates in a fashion that would be comparable in strength to sperm competition or overt male-male aggression is absent among Poecilia females, and (2) P. mexicana females typically form female-biased shoals, such that almost any female mate choice in nature occurs in front of a female audience. Poecilia females (P. mexicana, surface and cave form, and the closely related gynogenetic Poecilia formosa) were given a choice between a large and a small male, and the tests were repeated while a conspecific, a heterospecific, or no audience female (control) was presented. Females spent more time in the neutral zone and, thus, less time near the males during the second part of a trial when an audience was presented, but-consistent with predictions-females showed only slightly weaker expression of mate preferences during the second part of the tests. This decline was not specific to the treatment involving an audience and was significantly weaker than the effect seen in the male sex.
Orbiniid phylogeny is matter of debate and incongruence between hypothesis based on molecules and morphology has been repeatedly reported. Moreover, the phylogenetic position of the "oligochaetoid polychaetes" of the taxon Questa varies between morphological and molecular cladistic analyses. Here, we present a nearly complete mitochondrial genome of Questa ersei. The mitochondrial gene order is roughly identical to known orbiniid taxa. Several translocations of tRNAs are unique to Orbiniidae and Questa when compared to other annelid mitochondrial genomes. Additionally, we assembled sequence data of six genes (18S, 16S, cox1, cox3, nad1, nad4) for a representative orbiniid taxon sampling and analysed all data in concatenation using Maximum Likelihood and Bayesian inference. For comparison with morphology we compiled a morphological data matrix for all taxa included in our molecular analyses. Our results strongly support a close relationship of Questa with orbiniids (sequence data, gene order, an 18 bp indel, morphology), and a position nested within orbiniids is recovered in our sequence based analyses. We demonstrate remarkable incongruence of most included morphological characters with the recovered best ML tree and suppose that repeated independent character loss might be an explanation.
Using degenerate primers, we were able to identify seven Hox genes for the myzostomid Myzostoma cirriferum. The recovered fragments belong to anterior class (Mci_lab, Mci_pb), central class (Mci_Dfd, Mci_Lox5, Mci_Antp, Mci_Lox4), and posterior class (Mci_Post2) paralog groups. Orthology assignment was verified by phylogenetic analyses and presence of diagnostic regions in the homeodomain as well as flanking regions. The presence of Lox5, Lox4, and Post2 supports the inclusion of Myzostomida within Lophotrochozoa. We found signature residues within flanking regions of Lox5, which are also found in annelids, but not in Platyhelminthes. As such the available Hox genes data of myzostomids support an annelid relationship.
We analyzed mtDNA polymorphisms (parts of control region, ND5, ND2, Cytb, 12S, together 902 bp) in 59 scat and 18 tissue samples from 13 Indian populations of the critically endangered Indian tiger (Panthera tigris tigris), along with zoo animals as reference. Northern tiger populations exhibit two unique haplotypes suggesting genetic isolation. Western populations from Sariska (extinct in 2004) and Ranthambore are genetically similar, such that Ranthambore could serve as a source for reintroduction in Sariska. Zoo populations maintain mitochondrial lineages that are rare or absent in the wild.
It is well established that reproductive isolation often arises from genome incompatibilities and that genes encoding reproductive traits are less prone to introgression. Hybrid zones of Mytilus trossulus and Mytilus edulis provide an intriguing model to assess reproductive isolation. Although gene flow is restricted in North America, introgression is pervasive in the Baltic. This study aimed at analyzing the shape of multilocus clines across the Baltic contact zone between M. edulis and M. trossulus to infer mechanisms of restriction to gene flow. We use maximum likelihood methods to construct the best fitting individual clines for five markers located on biparentally inherited autosomes and paternally and maternally inherited mitochondrial DNA (mtDNA). Strong cline shape differences among markers suggest that reproductive isolation arising from genome-wide incompatibilities is weak, and that these discrepancies possibly result from genetic drift, hybrid zone movement or marker-specific selection. However, the finding of a common cline center for M7 lysin (involved in fertilization) and paternally transmitted mtDNA (causing nuclear-mitochondrial incompatibilities in hybrids) suggest that these loci may play a role in incomplete reproductive isolation.
Mate choice is mediated by a range of sensory cues, and assortative mating based on these cues can drive reproductive isolation among diverging populations. A specific feature of mormyrid fish, the electric organ discharge (EOD), is used for electrolocation and intraspecific communication. We hypothesized that the EOD also facilitates assortative mating and ultimately promotes prezygotic reproductive isolation in African weakly electric fishes. Our behavioural experiments using live males as well as EOD playback demonstrated that female mate recognition is influenced by EOD signals and that females are attracted to EOD characteristics of conspecific males. The dual function of the EOD for both foraging and social communication (including mate recognition leading to assortative mating) underlines the importance of electric signal differentiation for the divergence of African weakly electric fishes. Thus, the EOD provides an intriguing mechanism promoting trophic divergence and reproductive isolation between two closely related Campylomormyrus species occurring in sympatry in the lower Congo rapids.
Interlaminar differences of intrinsic properties of pyramidal neurons in the auditory cortex of mice
(2009)
Cortical information processing depends crucially upon intrinsic neuronal properties modulating a given synaptic input, in addition to integration of excitatory and inhibitory inputs. These intrinsic mechanisms are poorly understood in sensory cortex areas. We therefore investigated neuronal properties in slices of the auditory cortex (AC) of normal hearing mice using whole-cell patch-clamp recordings of pyramidal neurons in layers II/III, IV, V, and VI in the current- and voltage clamp mode. A total of 234 pyramidal neurons were included in the analysis revealing distinct laminar differences. Regular spiking (RS) neurons in layer II/III have significantly lower resting membrane potential, higher threshold for action potential generation, and larger K-ir and I-h amplitudes compared with layer V and VI RS neurons. These currents could improve temporal resolution in the upper layers of the AC. Additionally, the presence of a T-type Ca2+ current could be an important factor of RS neurons in these upper layers to amplify temporally closely correlated inputs. Compared with upper layers, lower layers (V and VI) exhibit a higher relative abundance of intrinsic bursting neurons. These neurons may provide layer-specific transfer functions for interlaminar, intercortical, and corticofugal information processing.
The centrosome is the main microtubule-organizing center and constitutes the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome is an established model for acentriolar centrosomes and it consists of a layered core structure Surrounded by a so-called corona, which harbors microtubule nucleation complexes. We have identified 34 new centrosomal candidate proteins through mass spectrometrical analysis of the proteome of isolated Dictyostelium centrosomes. Here we present a characterization of 12 centrosomal candidate proteins all featuring coiled coil regions and low expression levels, which are the most common attributes of centrosomal proteins. We used GFP fusion proteins to localize the candidate proteins in whole cells and on microtubule-free, isolated centrosomes. Thus we were able to identify nine new genuine centrosomal proteins including a putative orthologue of Cep192, an interaction partner of polo-like kinase 4 in human centriole biogenesis. In this respect, centrosomal localization of the only polo-like kinase in Dictyostelium, Pik, is also shown in this work. Using confocal deconvolution microscopy, four components, CP39, CP55, CP75, and CP91 could be clearly assigned to the so far almost uncharacterized centrosomal core structure, while CP148 and Cep192 localized to a zone between that of corona marker and core proteins. Finally, CP103 and CP248 were constituents of the corona. In contrast, NE81 was localized at the nuclear envelope and three others, an orthologue of the spindle checkpoint component Mad1, the novel Cenp68, and the centrosomal CP248 were observed at the centromeres, which are clustered and linked to the centrosome throughout the entire cell cycle. Cell Motil. Cytoskeleton 66: 915-928, 2009.
Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.
Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms, and generally involves Sun-family proteins located at the inner nuclear envelope. There is still no common scheme for the outer nuclear membrane proteins interacting with Sun I in centrosome/nucleus attachment. Here we propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium. For the first time we provide a detailed microscopic analysis of the centrosomal and nuclear envelope localization of endogenous Dictyostelium Sun1 during interphase and mitosis. By immunogold electron microscopy we show that Sun1 is a resident of both nuclear membranes. Disruption of Sun1 function by overexpression of full-length GFP-Sun1 or a GFP-Sun-domain deletion construct revealed not only the established function in centrosome/nucleus attachment and maintenance of ploidy, but also a requirement of Sun1 for the association of the centromere cluster with the centrosome. Live-cell imaging visualized the occurrence of mitotic defects, and demonstrated the requirement of microtubules for dynamic distance changes between centrosomes and nuclei. FRAP analysis revealed at least two populations of Sun1, with an immobile fraction associated with the centrosome, and a mobile fraction in the nuclear envelope.
Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium
(2009)
We analyzed relative sensitivities of small- and medium-sized carnivores to livestock husbandry (stocking rates and predator control) in Kalahari, South Africa, rangelands at a regional scale. We monitored small carnivores using track counts on 22 Kalahari farms across a land-use gradient ranging from low to high stocking rates and also interviewed each farm manager to identify farmers" perception of small carnivores as potential predators for livestock. We recorded 12 species of small- and medium-sized carnivores across 22 Kalahari farms. Stocking rate was the most important driving variable for local carnivore abundance. Abundance of all species was lowest on farms where stocking rate was high. Most farm managers perceived medium-sized carnivores, in particular, African wildcat (Felis silvestris lybica), black-backed jackal (Canis mesomelas), and caracal (Caracal caracal), as potential predators of livestock. Multiple regression analysis shows that black-backed jackal, African wildcat, and caracal were negatively affected by predator control measures, whereas bat-eared fox (Otocyon megalotis), cape fox (Vulpes chama), and small-spotted genet (Genetta genetta) were positively affected. Our results show a need for expanding research and conservation activities toward small- and medium-sized carnivores in southern African savannah rangelands. We, therefore, suggest developing a monitoring program combining passive tracking with indigenous knowledge of local Khoisan Bushmen to monitor carnivore populations, and we recommend additional predator removal experiments that manipulate predator densities.
Der Artikel liefert eine Zusammenstellung der aktuellen Ektoparasitenfauna der Fledermäuse (Flöhe, Fledermausfliegen, Wanzen, Zecken und Milben). Die Beziehung zwischen den Fledermäusen und ihren Ektoparasiten eignet sich in besonderem Maße als Modell ökologischer und evolutionärer Prozesse, wie das Überleben seltener Arten, die Kövolution zwischen Wirten und Parasiten, die Übertragung von Parasiten, die Wirtswahl und die Verbreitung von Krankheiten. Der gegenwärtige Stand der ökologischen Forschung auf diesen Gebieten wird zusammengefaßt und die Notwendigkeit und die Ziele weiterer Forschung werden angegeben.
This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.
While several authors suggest that bushbuck (Tragelaphus scriptus Pallas) from tropical areas with an approximately bimodal rainfall pattern breed throughout the year, there is also a report of seasonal breeding in this species. In this study, we provide indirect evidence of seasonality in reproduction by analysing behavioural data (e.g. rates of mixed-sex sightings) in a population of bushbuck inhabiting an equatorial savannah ecosystem in western Uganda. Observation rates of mixed-sex sightings were correlated with rainfall patterns. We suggest that peaks in reproductive behaviour following the wet season may be advantageous if calves are born during the next wet season, when fresh vegetation is available.
Savannah areas affected by human activities such as livestock keeping and agriculture are often characterized by shifts in landscape structuring, with a predominance of few(er) habitat types. This is typically accompanied by pronounced changes in the communities of ungulates. The aim of this study was to find out whether shifts in ungulate communities in Lake Mburo National Park (LMNP) are primarily predicted by an alteration in the composition of the preferred habitat types or if more complex interactions between habitat changes and the prevalence of ungulates occur. Monthly road counts were used to establish the number of eleven ungulate species in LMNP and adjacent unprotected Ankole Ranching Scheme. The common duiker (Sylvicapra grimmia campbelliae Gray, 1843) was found in more abundance in disturbed areas, while showing a significant change in habitat use. Common duiker tended to use the vegetation type otherwise used by the bushbuck (Tragelaphus scriptus dama Neumann, 1902). Our results support the claim that the occurrence of ungulates is not only directly affected by the availability of 'suitable' habitats, but behavioural plasticity and competitive exclusion also need to be considered.
We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.
Dry lands are exposed to a highly variable environment and face a high risk of degradation. The effects of climate change are likely to increase this risk; thus a profound knowledge of the system dynamics is crucial for evaluating management options. This applies particularly for the interactions between water and vegetation, which exhibit strong feedbacks. To evaluate these feedbacks and the effects of climate change on soil moisture dynamics, we developed a generic, process-based, spatially explicit soil moisture model of two soil layers, which can be coupled with vegetation models. A time scale relevant for ecological processes can be simulated without difficulty, and the model avoids complex parameterization with data that are unavailable for most regions of the world. We applied the model to four sites in Israel along a precipitation and soil type gradient and assessed the effects of climate change by comparing possible climatic changes with present climate conditions. The results show that in addition to temperature, the total amount of precipitation and its intra-annual variability are an important driver of soil moisture patterns. This indicates that particularly with regard to climate change, the approach of many ecological models that simulate water dynamics on an annual base is far too simple to make reliable predictions. Thus, the introduced model can serve as a valuable tool to improve present ecological models of dry lands because of its focus on the applicability and transferability.
The new species Erysiphe asclepiadis is described, illustrated and discussed. A new Chinese collection of Erysiphe robiniicola has recently been found that can be used to elucidate and discuss the confused taxonomy and nomenclature of this species and other taxa of Erysiphe s. lat. on Robinia spp. Based on a re-examination of type material in connection with the data given in the protologue, it can be shown that Capnodium lygodesmiae must be reduced to synonymy with Ampelomyces quisqualis. The confusion surrounding the name C. lygodesmiae, caused by the occurrence of the hyperparasite A. quisqualis on a powdery mildew fungus with abundant chasmothecia, is discussed in detail. The new combination, Golovinomyces caulicola (; Spolverinia caulicola), is proposed for the powdery mildew that serves as host of C. lygodesmiae.
In common garden experiments, a number of genotypes are raised in a common environment in order to quantify the genetic component of phenotypic variation. Common gardens are thus ideally suited for disentangling how genetic and environmental factors contribute to the success of invasive species in their new non-native range. Although common garden experiments are increasingly employed in the study of invasive species, there has been little discussion about how these experiments should be designed for greatest utility. We argue that this has delayed progress in developing a general theory of invasion biology. We suggest a minimum optimal design (MOD) for common garden studies that target the ecological and evolutionary processes leading to phenotypic differentiation between native and invasive ranges. This involves four elements: (A) multiple, strategically sited garden locations, involving at the very least four gardens (2 in the native range and 2 in the invaded range); (B) careful consideration of the genetic design of the experiment; (C) standardization of experimental protocols across all gardens; and (D) care to ensure the biosafety of the experiment. Our understanding of the evolutionary ecology of biological invasions will be greatly enhanced by common garden studies, if and only if they are designed in a more systematic fashion, incorporating at the very least the MOD suggested here.
We investigated sex-specific parental care behaviour of lesser spotted woodpeckers Picoides minor in the low mountain range Taunus, Germany. Observed parental care included incubation, nest sanitation as well as brooding and feeding of nestlings. Contributions of the two sexes to parental care changed in progress of the breeding period. During incubation and the first half of the nestling period, parental care was divided equally between partners. However, in the late nestling stage, we found males to feed their nestlings irrespective of brood size while females considerably decreased feeding rate with the number of nestlings. This behaviour culminated in desertion of small broods by females shortly before fledging. The fact that even deserted nests were successful indicates that males were able to compensate for the females' absence. Interestingly, the mating of one female with two males with separate nests could be found in the population, which confirms earlier findings of polyandry in the lesser spotted woodpecker. We conclude that biparental care is not essential in the later stage and one partner can reduce effort and thus costs of parental care, at least in small broods where the mate is able to compensate for that behaviour. Reduced care and desertion appears only in females, which might be caused by a combination of two traits: First, females might suffer higher costs of investment in terms of mortality and secondly, male-biased sex ratio in the population generally leads to higher mating probabilities for females in the following breeding season. The occurrence of polyandry seems to be a result of these conditions.
A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare) termed cytoplasmic line 3 (CL3), displaying a viridis (homogeneously light-green colored) phenotype, has been previously shown to be affected by elevated temperatures. In this article, biochemical, biophysical, and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I (PSI) under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII.
The layer-by-layer adsorption technique based on the consecutive deposition of oppositely charged species is for the preparation of protein multilayers with fully electro-active protein molecules. The methodology was established with cytochrome c and the polyelectrolyte sulfonated polyaniline (PASA). The technique is also useful for the construction of bi-protein architectures confining protein-protein communication to an electrode. Following natural examples of protein complexes with defined signal transfer, cytochrome c was arranged with enzymes such as xanthine oxidase, bilirubin oxidase, laccase, and sulfite oxidase in self-assembled multilayer architectures. Thus, biomimetic signal chains from the enzyme substrate via the enzyme and cytochrome c towards the electrode can be established. Communication between proteins immobilised in multiple layers on the electrode can be achieved by in situ generation of small shuttle molecules or more advantageously by direct interprotein electron transfer. This allows the construction of new sensing electrodes, the properties of which can be tuned by the number of deposited protein layers. The mechanism of electron transfer within such protein assemblies on gold electrodes will be discussed.
Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase
(2009)
In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples (OsHRP)-H-III/(OsHRP)-H-IV, (OsHRP)-H-IV/(OsHRP)-H-V and (OsHRP)-H-V/ (OsHRP)-H-VI. The midpoint potentials differ dependent on the electrode material used with E-1/2 (Os-III/IV) of -0.4 V (ITO) and -0.25 V (GC), E-1/2 (Os-IV/V) of -0.16 V (ITO) and +0.10 V (GC), and E-1/2 (Os-V/VI)of +018 V (ITO), respectively Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.
We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca2+-dependent Cl- current (I-CLCA). Here we report that I-CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al3+-sensitive, voltage-dependent, Ca2+ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (-60 mV), I-CLCA was found to be inactive and resting currents were predominantly K+ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I-CLCA (T (0.5) similar to 500 s), while repolarization in turn resulted in a monoexponential decay in I-CLCA (T (0.5) similar to 100 s). The activation of I-CLCA by depolarization was reduced by lowering extracellular Ca2+ and completely inhibited by buffering cytosolic Ca2+ with EGTA, suggesting a role for Ca2+ influx in the activation of I-CLCA. However, raising bulk cytosolic Ca2+ at -60 mV did not produce sustained I-CLCA activity. Therefore I-CLCA is dependent on both an increase in intracellular Ca2+ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca2+ influx pathway that displays equal permeability to Ca2+ and Ba2+ ions and that is blocked by extracellular Al3+ and La3+. Furthermore, Al3+ completely and reversibly inhibited depolarization-induced activation of I-CLCA, thereby directly linking Ca2+ influx to activation of I-CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I-CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.
Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.
We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 +/- 0.01 min(-1) was obtained. A1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 +/- 0.02 mu M for CTP and 1.17 +/- 0.18 mu M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.
Die in der Umgebung von potsdam untersuchten Nester der Mehlschwalbe (Delichon urbica) enthielten drei spezifische Ektoparasitenarten in unterschiedlicher Dichte. Pro nest fanden sich von der Mehlschwalbenlausfliege (Stenepteryx hirundinis) 0-12 Eier, von der Mehlschwalbenwanze (Oeciacus hirundinis)195-1104 Larven und Imagines und vom Mehlschwalbenfloh (Ceratophyllus hirundinis)9-135 Imagines. Zwischen 1. Oktober und dem 9.-16. mai des folgenden Jahres gab es im Durchschnitt nur einen geringfuegigen Rueckgang der Anzahl der Wanzen, waehrend die Abundanz adulter Floehe deutlich zunahm. Die Parasitendichte schwankte zwischen verschiedenen Fundorten und selbst innerhalb einer Nestreihe erheblich. In den am staerksten mit Parasiten belasteten Nestern ist eine erfolgreiche Aufzucht der Jungtiere durch die Mehlschwalben unwahrscheinlich. Die Hoehe der Parasitenlast koennte die Wahl der Schwalbenzwischen der Nutzung vorjaehriger Nester oder dem Ausbau aelterer nester wesentlich beeinflussen.
Preschool age is a biological stage of intensive longitudinal growth with high plasticity of the growing body and of body postures. It is the period where children learn to persist in a sitting posture for a longer time and to use furniture like chairs or other body supporting systems. The growing body shows a special sensitivity for the manifestation of inappropriate postures. In this study the development of body measurements and sitting behaviour of preschool age children is investigated as a precondition for an optimal adjustment of seats and desks to the growing body. Accordingly to the instructions of Knußmann (1988) and Jürgens (1988) 6 body measurements were taken from 122 German children aged 3 to 7 years from Potsdam, Province Brandenburg. Additionally, every child was videotaped for 10 minutes while crayoning in a sitting position of its own choice using a chair and a desk. To analyse the tapes, the software Noldus Observer was used and examined, picture by picture, to define the different types of sitting postures as well as the duration of persistence in a posture and the number of changes of postures. The used chairs and desks were also measured. Furthermore, the data of the furniture guideline DIN ISO 5970 (DIN, 1981), which regulates the dimensions of furniture for sitting in educational institutions, were compared with the results of the body measurements and with the dimensions of the furniture used by the children.
Modelling ungulate dependence on higher quality forage under large trees in African savannahs
(2009)
In African savannahs, large trees improve grass quality, particularly in dry and nutrient poor areas. Enhanced below-canopy grass nutrients, such as nitrogen and phosphorus contents should therefore attract and benefit grazers. To predict whether ungulates really need these forage quality islands we focused on four grazer species, i.e., zebra, buffalo, wildebeest, and warthog, differing in body size and digestive system. We confronted literature estimations of their feeding requirements with forage availability and quality, observed in three South African savannah systems, through linear modelling. The model predicted the proportion of below-canopy grass that grazers should include in their diet to meet their nutritional requirements. During the wet season, the model predicted that all animals could satisfy their daily nutrient requirements when feeding on a combination of below- and outside-canopy grasses. However, wildebeest, having relatively high nutrient demands, could meet their nutrient requirements only by feeding almost exclusively below canopies. During the dry season, all animals could gain almost twice as much digestible protein when feeding on below - compared to outside-canopy forage. Nonetheless, only warthogs could satisfy their nutrient requirements - when feeding almost exclusively on below-canopy grasses. The other ungulate species could not meet their phosphorus demands by feeding at either site without exceeding their maximum fibre intake, indicating the unfavourable conditions during the dry season. We conclude that grazing ungulates, particularly warthog, zebra, and buffalo, actually depend on the available below-canopy grass resources. Our model therefore helps to quantify the importance of higher quality forage patches beneath savannah trees. The composition of grazer communities depending on below-canopy grasses can be anticipated if grazer food requirements and the abundance of large trees in savannahs are known. The model suggests that the conservation of large single-standing trees in savannahs is crucial for maintenance of locally grazing herbivores.
Genetic structure and dispersal in a small South African Rodent : is dispersal female-biased?
(2009)
Dispersal greatly determines genetic structure of populations, although it is influenced by landscape heterogeneity, quality of the matrix, resource distribution and local population densities and dynamics. To get insights into some of those processes we analysed the genetic structure of the hairy-footed gerbil Gerbillurus paeba (Rodentia, Murinae, Gerbillinae) in the southern Kalahari (South Africa). Samples were taken from 20 populations covering an area of about 2200 km2. Genetic data were related to landscape characters and population dynamics. We used newly developed microsatellites and found at all loci some indication for the presence of null alleles. However, null alleles seem to have little influence on the general results of our analyses. Altogether we found even nearby populations of G. paeba to be significantly differentiated, although assignment tests revealed 24% of individuals as immigrants. Genetic structure was independent of landscape heterogeneities at all spatial scales. Autocorrelation analyses (range 50-90 km) revealed significant genetic structure within populations on distances <3 km. We found some indication for female-biased dispersal. Our study suggests that dispersing individuals have little influence on the long-term genetic structure and that drift is the major cause of genetic diversity. The observed genetic pattern likely derives from strong population fluctuations of G. paeba. The landscape structure has little influence on the genetic differentiation between populations.
Rhodococcus fascians is a Gram-positive phytopathogen that induces shooty hyperplasia on its hosts through the secretion of cytokinins. Global transcriptomics using microarrays combined with profiling of primary metabolites on infected Arabidopsis (Arabidopsis thaliana) plants revealed that this actinomycete modulated pathways to convert its host into a niche. The transcript data demonstrated that R. fascians leaves a very characteristic mark on Arabidopsis with a pronounced cytokinin response illustrated by the activation of cytokinin perception, signal transduction, and homeostasis. The microarray data further suggested active suppression of an oxidative burst during the R. fascians pathology, and comparison with publicly available transcript data sets implied a central role for auxin in the prevention of plant defense activation. Gene Ontology categorization of the differentially expressed genes hinted at a significant impact of infection on the primary metabolism of the host, which was confirmed by subsequent metabolite profiling. The much higher levels of sugars and amino acids in infected plants are presumably accessed by the bacteria as carbon and nitrogen sources to support epiphytic and endophytic colonization. Hexoses, accumulating from a significantly increased invertase activity, possibly inhibited the expression of photosynthesis genes and photosynthetic activity in infected leaves. Altogether, these changes are indicative of sink development in symptomatic tissues. The metabolomics data furthermore point to the possible occurrence of secondary signaling during the interaction, which might contribute to symptom development. These data are placed in the context of regulation of bacterial virulence gene expression, suppression of defense, infection phenotype, and niche establishment.
Phage tailspike proteins with beta-solenoid fold as thermostable carbohydrate binding materials
(2009)
We have investigated the stability of three tailspike proteins (TSPs) from bacteriophages Sf6, P22, and HK620. Tailspikes are rod-like homotrimers with comparable beta-solenoid folds and similarly high kinetic stability in spite of different amino acid sequences. As tailspikes bind polysaccharides to recognize the bacterial host cell, their stability is required for maintenance of bacteriophage infectivity under harsh extracellular conditions. They resist denaturation by SDS at ambient temperature and their unfolding is slow even in 6 m guanidinium hydrochloride (GdmHCl). This makes them interesting candidates for very stable carbohydrate binding protein materials.
Kinetic modelling of complex metabolic networks - a central goal of computational systems biology - is currently hampered by the lack of reliable rate equations for the majority of the underlying biochemical reactions and membrane transporters. On the basis of biochemically substantiated evidence that metabolic control is exerted by a narrow set of key regulatory enzymes, we propose here a hybrid modelling approach in which only the central regulatory enzymes are described by detailed mechanistic rate equations, and the majority of enzymes are approximated by simplified (nonmechanistic) rate equations (e.g. mass action, LinLog, Michaelis-Menten and power law) capturing only a few basic kinetic features and hence containing only a small number of parameters to be experimentally determined. To check the reliability of this approach, we have applied it to two different metabolic networks, the energy and redox metabolism of red blood cells, and the purine metabolism of hepatocytes, using in both cases available comprehensive mechanistic models as reference standards. Identification of the central regulatory enzymes was performed by employing only information on network topology and the metabolic data for a single reference state of the network [Grimbs S, Selbig J, Bulik S, Holzhutter HG & Steuer R (2007) Mol Syst Biol3, 146, doi:10.1038/msb4100186]. Calculations of stationary and temporary states under various physiological challenges demonstrate the good performance of the hybrid models. We propose the hybrid modelling approach as a means to speed up the development of reliable kinetic models for complex metabolic networks.
Genome-scale metabolic networks which have been automatically derived through sequence comparison techniques are necessarily incomplete. We propose a strategy that incorporates genomic sequence data and metabolite profiles into modeling approaches to arrive at improved gene annotations and more complete genome-scale metabolic networks. The core of our strategy is an algorithm that computes minimal sets of reactions by which a draft network has to be extended in order to be consistent with experimental observations. A particular strength of our approach is that alternative possibilities are suggested and thus experimentally testable hypotheses are produced. We carefully evaluate our strategy on the well-studied metabolic network of Escherichia coli, demonstrating how the predictions can be improved by incorporating sequence data. Subsequently, we apply our method to the recently sequenced green alga Chlamydomonas reinhardtii. We suggest specific genes in the genome of Chlamydomonas which are the strongest candidates for coding the responsible enzymes.
Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase
(2009)
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6- aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.
Gating of K+ and other ion channels is 'hard-wired' within the channel protein. So it remains a puzzle how closely related channels in plants can show an unusually diverse range of biophysical properties. Gating of these channels lies at the heart of K+ mineral nutrition, signalling, abiotic and biotic stress responses in plants. Thus, our knowledge of the molecular mechanics underpinning K+ channel gating will be important for rational engineering of related traits in agricultural crops. Several key studies have added significantly to our understanding of channel gating in plants and have challenged current thinking about analogous processes found in animal K+ channels. Such studies highlight how much of K+ channel gating remains to be explored in plants.
Occludin is a self-associating transmembrane tight junction protein affected in oxidative stress. However, its function is unknown. The cytosolic C-terminal tail contains a coiled coil-domain forming dimers contributing to the self- association. Studying the hypothesis that the self-association is redox-sensitive, we found that the dimerization of the domain depended on the sulfhydryl concentration of the environment in low-millimolar range. Under physiological conditions, monomers and dimers were detected. Masking the sulfhydryl residues in the domain prevented the dimerization but affected neither its helical structure nor cylindric shape. Incubation of cell extracts containing full-length occludin with sulfhydryl reagents prevented the dimerization; a cysteine/alanine exchange mutant also did not show dimer formation. This demonstrates, for the first time, that disulfide bridge formation of the domain is involved in the occludin dimerization. It is concluded that the redox-dependent dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions.
We studied the effects of overgrazing on the foraging behaviour of the lizard Pedioplanis l. lineoocellata (Spotted Sand Lizard), a sit-and-wait forager, in habitats of differing vegetation states to determine the effects of habitat degradation on this species. At high grazing intensity where vegetation cover and diversity is low, the lizard P. lineoocellata moves more frequently, spends more time moving and covers larger distances than in habitats where vegetation cover and diversity is high. These behavioural changes in movement patterns can be explained by less abundant prey in habitats with low vegetation cover and diversity. Although morphology, phylogeny and physiology of P. lineoocellata should constrain the change in foraging behaviour, the species has modified its foraging strategy from sit- and-wait to actively foraging. We assume that this behavioural flexibility of P. lineoocellata is a buffer mechanism enabling the species to use and survive in degraded (unfavourable) habitats.
Exceeding weight gain in childhood is a prevailing issue in industrialised countries, such as in Germany. The aim of this study was to detect a critical age for exceeding weight gain. It is assumed that especially in the early years of life, the years of nursery school age, the individual development of weight is fundamental for the prediction of obesity. The data of 638 children (324 boys and 314 girls) and the data of additional 1390 children of a preceding longitudinal study were analysed. The results show that overweight newborns are not at higher risk of becoming overweight children later, in the first place. But the results identify a high risk of becoming overweight for children 4.5 years old through a BMI rebound. In addition, this comparably earlier BMI rebound is linked with an increasing percentage of body fat. This leads to the assumption, that a comparably early BMI rebound is remarkably atypical for healthy child development. The results are also interrelated with the test person's sex. Endangered girls are of pyknomorphic body type. In contrast, the boys' results are independent of the body type. Obesity of boys therefore is strongly assumed to be caused by environmental factors.
Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 A mu l whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins.
The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV-vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.
Activated carbon has become a widely used tool to investigate root-mediated allelopathy of plants, especially in plant invasion biology, because it adsorbs and thereby neutralizes root exudates. Allelopathy has been a controversially debated phenomenon for years, which revived in plant invasion biology as one possible reason for the success of invasive plants. Noxious plant exudates may harm other plants and provide an advantage to the allelopathic plant. However, root exudates are not always toxic, but may stimulate the microbial community and change nutrient availability in the rhizosphere. In a greenhouse experiment, we investigated the interacting effects of activated carbon, arbuscular mycorrhiza and plant competition between the invasive Senecio inaequidens and the native Artemisia vulgaris. Furthermore, we tested whether activated carbon showed any undesired effects by directly affecting mycorrhiza or soil chemistry. Contrary to the expectation, S. inaequidens was a weak competitor and we could not support the idea that allelopathy was involved in the competition. Activated carbon led to a considerable increase in the aboveground biomass production and reduced the infection with arbuscular mycorrhiza of both plant species. We expected that arbuscular mycorrhiza promotes plant growth by increasing nutrient availability, but we found the contrary when activated carbon was added. Chemical analyses of the substrate showed, that adding activated carbon resulted in a strong increase in plant available phosphate and in a decrease of the C-organic/N-total ration both of which suggest stimulated microbial activity. Thus, activated carbon not only reduced potential allelopathic effects, but substantially changed the chemistry of the substrate. These results show that activated carbon should be handled with great care in ecological experiments on allelopathy because of possible confounding effects on the soil community.
Tree size and herbivory determine below-canopy grass quality and species composition in savannahs
(2009)
Large single-standing trees are rapidly declining in savannahs, ecosystems supporting a high diversity of large herbivorous mammals. Savannah trees are important as they support both a unique flora and fauna. The herbaceous layer in particular responds to the structural and functional properties of a tree. As shrubland expands stem thickening occurs and large trees are replaced by smaller trees. Here we examine whether small trees are as effective in providing advantages for grasses growing beneath their crowns as large trees are. The role of herbivory in this positive tree- grass interaction is also investigated. We assessed soil and grass nutrient content, structural properties, and herbaceous species composition beneath trees of three size classes and under two grazing regimes in a South African savannah. We found that grass leaf content (N and P) beneath the crowns of particularly large (ca. 3.5 m) and very large trees (ca. 9 m) was as much as 40% greater than the same grass species not growing under a tree canopy, whereas nutrient contents of grasses did not differ beneath small trees (< 2.3 m). Moderate herbivory enhanced these effects slightly. Grass species composition differed beneath and beyond the tree canopy but not between tree size classes. As large trees significantly improve the grass nutrient quality for grazers in contrast to smaller trees, the decline of the former should be halted. The presence of trees further increases grass species diversity and patchiness by favouring shade- tolerant species. Both grazing wildlife and livestock will benefit from the presence of large trees because of their structural and functional importance for savannahs.
We investigated the response of two populations of the barb Garra barreimiae to different light intensities (0.5-2000 lx) from a light source. Adults of both the surface (epigean) and cave (hypogean) G. barreimiae populations show photophobic behavior. A photophobic response in the cave form was seen only at higher light intensities because the cavefish are eyeless and rely on extra-retinal light receptors to detect light. In contrast, juveniles (surface and cave) showed photophilic behavior, and their preference for the photic zone of the test tank decreased with increasing age. We discuss the potential role played by photophobic behavior for the colonization of caves by previously surface- dwelling fishes.
Terrestrial-derived dissolved organic carbon (DOC) contributes significantly to the energetic basis of many aquatic food webs. Although heterotrophic bacteria are generally considered to be the sole consumers of DOC, algae and cyanobacteria of various taxonomic groups are also capable of exploiting this resource. We tested the hypothesis that algae can utilise DOC in the presence of bacteria if organic resources are supplied in intervals by photolysis of recalcitrant DOC. In short-term uptake experiments, we changed irradiation in the range of minutes. As model substrates, polymers of radiolabelled coumaric acid (PCA) were used, which during photolysis are known to release aromatic compounds comparable to terrestrial-derived and refractory DOC. Three cultured freshwater algae readily assimilated PCA photoproducts equivalent to a biomass-specific uptake of 5-60% of the bacterial competitors present. Algal substrate acquisition did not depend on whether PCA was photolysed continuously or in intervals. However, the data show that photoproducts of terrestrial DOC can be a significant resource for osmotrophic algae. In long-term growth experiments, interval light was applied one hour per day. We allowed cultured Chlamydomonas to compete for ambient DOC of low concentration. We found higher abundances of Chlamydomonas when cultures were irradiated intermittently rather than continuously. These data suggest that photolysis of DOC supports algal heterotrophy, and potentially facilitates growth, when light fluctuations are large, as during the diurnal light cycle. We concluded that osmotrophic algae can efficiently convert terrestrial carbon into the biomass of larger organisms of aquatic food webs.
The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.
The yellowhammer Emberiza citrinella is a common European bird that sings in dialects that for decades have been distinguished by the existence of one single element (called a "specific''). In this study we looked into other possibilities for dialect discrimination, measuring 24 different variables. For the first time, multivariate statistics were used to discriminate dialect in yellowhammer song. Two similar dialects (XlB and XsB) that are not clearly defined in the literature were studied. Statistics incorporated (1) all variables, ( 2) no variables of "specific'' elements, and (3) no variables under the influence of these "specific'' variables. Multivariate statistics support dialect discrimination by ear and confirmed that only one element in yellowhammer song characterises dialect. In addition, we looked for local differences within two dialects and found that one local observation area showed a higher separation than the other sites (Meck1). However, as yet there is insufficient evidence for the existence of a new subdialect.
It has been suggested that all species of spiral-horned antelopes (Tragelaphini) lack territoriality. Furthermore, some authors suggested that bushbuck (Tragelpahus scriptus) males form dominance hierarchies. In this study, we investigated the dominance relationships in two groups of free-ranging bushbuck males in Queen Elizabeth National Park, Uganda. Adult males dominated young-adult bachelors and subadult males, but no distinct dominance relationships were found among adult males. Landau's index of linearity revealed no linear dominance hierarchy in the study populations. Our results support the idea that adult males are territorial, and overt aggression is directed almost exclusively towards bachelors that challenge territory holders.
Grouping patterns within the genus Tragelaphus suggest that species inhabiting open areas tend to live in larger groups, while species preferring dense habitats live solitarily or in small family groups. We asked if similar variation would be concealed in the within-species variation of bushbuck (Tragelaphus scriptus). Recent molecular phylogeographic analyses revealed several locally adapted forms of bushbuck in different ecoregions on the African continent. We compared group sizes of south-eastern bushbuck (Tragelaphus sylvaticus) among six different populations ("ecotypes"). To date, most data on the social organization of bushbuck have been collected from only one population in Queen Elizabeth National Park (QENP) in Uganda. This particular population, however, inhabits an unusual-comparatively open-habitat type, while bushbuck otherwise inhabit dense habitats, leaving doubt whether data collected in QENP are representative of the entire species. We, therefore, compared grouping patterns between ecotypes inhabiting rather open habitats (e.g., dianae and haywoodi) and ecotypes occupying rather dense habitats (e.g., massaicus and ornatus). In bachelor groups and in all-female (spinster) groups, single sightings were the most frequent "group type" in all populations examined. We detected no significant difference among ecotypes in relative frequencies of group size categories in the case of bachelor groups. Spinster group sizes were slightly (albeit significantly) smaller in QENP than in all other areas. Moreover, a comparison of two areas inside (low human pursuit) and outside Lake Mburo National Park (high hunting pressure) in Uganda revealed no significant difference in grouping patterns in response to human pursuit (as reported for impala [Aepyceros melampus] inhabiting the same area). Altogether, our results suggest that group sizes in bushbuck are not dependent on the habitat type they inhabit; neither does human nuisance have an impact on grouping patterns. Hence, an "almost solitary" lifestyle appears to be a characteristic of the entire taxon.
Background and purpose: Although carbon monoxide (CO) can modulate inflammatory processes, the influence of CO on adhesion molecules is less clear. This might be due to the limited amount of CO generated by haem degradation. We therefore tested the ability of a CO releasing molecule (CORM-3), used in supra-physiological concentrations, to modulate the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on endothelial cells and the mechanism(s) involved. Experimental approach: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor (TNF)-alpha in the presence or absence of CORM-3. The influence of CORM-3 on VCAM-1 and E- selectin expression and the nuclear factor (NF)-kappa B pathway was assessed by flow cytometry, Western blotting and electrophoretic mobility shift assay. Key results: CORM-3 inhibited the expression of VCAM-1 and E-selectin on TNF-alpha- stimulated HUVEC. VCAM-1 expression was also inhibited when CORM-3 was added 24 h after TNF-alpha stimulation or when TNF-alpha was removed. This was paralleled by deactivation of NF-kappa B and a reduction in VCAM-1 mRNA. Although TNF- alpha removal was more effective in this regard, VCAM-1 protein was down-regulated more rapidly when CORM-3 was added. CORM-3 induced haem oxygenase-1 (HO-1) in a dose- and time-dependent manner, mediated by the transcription factor, Nrf2. CORM-3 was still able to down-regulate VCAM-1 expression in HUVEC transfected with siRNA for HO-1 or Nrf2. Conclusions and implications: Down-regulation of VCAM and E-selectin expression induced by CORM-3 was independent of HO-1 up- regulation and was predominantly due to inhibition of sustained NF-kappa B activation.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
Peripheral T-cell (TC) tolerance can be induced by tolerogenic antigen-presenting cell (APC). A prerequisite is the reduction or blockade of B7 of APC. Besides dendritic cell, B cells can be used as APC. Here, we show the generation B cells with reduced B7 expression by lentiviral transduction of endoplasmic reticulum (ER)-directed CTLA4. Vectors coding for the human CTL4-Ig were used for the human B-cell line Raji. Transduction efficiency was over 90% (MOI = 3). For the murine B-cell line A20 and for primary mouse B cells, murine CTLA4 was used. We show that B cells with reduced B7 expression reduce the antigen (Ag) specific TC proliferation in vitro. B cells expressing an ER-directed CTLA4 may reduce Ag-specific immune responses.
Secretion in blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT), which activates the InsP(3)/Ca2+ pathway and the cAMP/protein kinase A (PKA) pathway in the secretory cells. The latter signaling cascade induces the activation of a vacuolar H+-ATPase on the apical membrane. Here, we have determined the distribution of PKA by using antibodies against the PKA regulatory subunit-II (PKA-RII) and the PKA catalytic subunit (PKA-C) of Drosophila. PKA is present in high concentrations within the secretory cells. PKA-RII and PKA-C co-distribute in non-stimulated glands, being enriched in the basal portion of the secretory cells. Exposure to 8-CPT-cAMP or 5-HT induces the translocation of PKA-C to the apical membrane, whereas the PKA-RII distribution remains unchanged. The recruitment of PKA-C to the apical membrane corroborates our hypothesis that vacuolar H+-ATPase, which is enriched in this membrane domain, is a target protein for PKA.
Occludin is a self-associating transmembrane tight junction protein affected in oxidative stress. However, its function is unknown. The cytosolic C-terminal tail contains a coiled coil-domain forming dimers contributing to the self- association. Studying the hypothesis that the self-association is redox-sensitive, we found that the dimerization of the domain depended on the sulfhydryl concentration of the environment in low-millimolar range. Under physiological conditions, monomers and dimers were detected. Masking the sulfhydryl residues in the domain prevented the dimerization but affected neither its helical structure nor cylindric shape. Incubation of cell extracts containing full-length occludin with sulfhydryl reagents prevented the dimerization; a cysteine/alanine exchange mutant also did not show dimer formation. This demonstrates, for the first time, that disulfide bridge formation of the domain is involved in the occludin dimerization. It is concluded that the redox-dependent dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions.
The transmembrane tight junction protein occludin is sensitive to oxidative stress. Occludin oligomerizes; however, its function in the tight junction is unknown. The cytosolic C-terminal tail contains a coiled coil-domain and forms dimers contributing to the oligomerization. The regulation of the oligomerization remains unclear. As the domain area contains sulfhydryl residues, we tested the hypothesis that the dimerization of the coiled coil-domain depends on these residues. We showed that the dimerization is modulated by the thiol concentration in the low-millimolar range, which is relevant both for physiological and pathophysiological conditions. Masking the sulfhydryl residues in the fragment by covalent binding of 4-vinyl pyridine prevented the dimerization but did not affect its helical structure and cylindric shape. The data demonstrate, for the first time, that disulfide bridge formation of murine cystein 408 is involved in the dimerization. This process is redox-sensitive but the secondary structure of the domain is not. It is concluded that the dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions.
Plant communities can be affected both by arbuscular mycorrhizal fungi (AMF) and hemiparasitic plants. However, little is known about the interactive effects of these two biotic factors on the productivity and diversity of plant communities. To address this question, we set up a greenhouse study in which different AMF inocula and a hemiparasitic plant (Rhinanthus minor) were added to experimental grassland communities in a fully factorial design. In addition, single plants of each species in the grassland community were grown with the same treatments to distinguish direct AMF effects from indirect effects via plant competition. We found that AMF changed plant community structure by influencing the plant species differently. At the community level, AMF decreased the productivity by 15-24%, depending on the particular AMF treatment, mainly because two dominant species, Holcus lanatus and Plantago lanceolata, showed a negative mycorrhizal dependency. Concomitantly, plant diversity increased due to AMF inoculation and was highest in the treatment with a combination of two commercial AM strains. AMF had a positive effect on growth of the hemiparasite, and thereby induced a negative impact of the hemiparasite on host plant biomass which was not found in non-inoculated communities. However, the hemiparasite did not increase plant diversity. Our results highlight the importance of interactions with soil microbes for plant community structure and that these indirect effects can vary among AMF treatments. We conclude that mutualistic interactions with AMF, but not antagonistic interactions with a root hemiparasite, promote plant diversity in this grassland community.
Die in Deutschland gegenwärtig durch Nährstoffeinträge und ausbleibenden Nährstoffentzug stark im Rückgang begriffenen Flechten-Kiefernwälder werden als Biotoptyp wie auch als Lebensraumtyp "Mitteleuropäische Flechten-Kiefernwälder" (Code 91T0) diskutiert. Die bisherige, sehr uneinheitliche Differenzierung von Flechten-Kiefernwäldern auf der Ebene von Biotoptypen wird dargestellt. Auf der Grundlage neuerer vegetationskundlicher übersichten werden Vorschläge für eine einheitliche Abgrenzung des Biotoptyps "Flechten-Kiefernwald" und des Lebensraumtyps 91T0 unterbreitet. Im niedersächsischen Naturwaldreservat "Kaarßer Sandberge" (Niedersachsen) wurde die Anwendung des Konzeptes erfolgreich erprobt. Nicht nur hier, sondern auch deutschlandweit wird der Rückgang der Erdflechten in den Kieferwäldern zugunsten von Drahtschmiele und/ oder pleurokarpen Moosen deutlich. Nach der derzeitigen Definition des Lebensraumtyps 91T0 besteht auf der Grundlage der FFH-Richtlinie nicht für alle Flechten-Kiefernwälder eine Chance der Verbesserung. Der Ausschluss von außerhalb des natürlichen Verbreitungsgebietes der Wald-Kiefer gelegenen sowie von durch Aufforstung angepflanzten Beständen bringt Probleme mit sich, die diskutiert werden. Für den Erhalt und die Wiederherstellung der größtenteils nutzungsbedingt entstandenen Flechten-Kiefernwälder sind praktikable Pflegemaßnahmen notwendig, die im Rahmen von Streunutzungsversuchen erprobt werden müssen.
Besides habitat loss, population-biological and genetic consequences of habitat fragmentation are thought to be a major threat to species since the 1990's and thus are now in the focus of plant species conservation. Using examples, this article gives an overview on the state of the art. It aims to evaluate the relevance habitat fragmentation and the resulting small size and isolation of populations may have for Central European plant populations. Stochasticity, edge effects, pollinator limitation, genetic drift and inbreeding depression are identified as important and very widespread negative effects. Together with changed habitat quality due to eutrophication, drainage or altered land use they negatively affect the fitness of individuals and populations, resulting in an increased risk of extinction. This negative effect of small populations on the fitness of individuals is called the Allee-effect, irrespective of the underlying causes, which can only be identified by scientific experiments. Metapopulation dynamics that are supported by a habitat network may prevent a permanent extinction of plant populations and minimize the negative genetic effects of habitat fragmentation by increasing gene flow via pollen and seeds. However, existing studies from Central Europe mainly concentrated on certain plant families (Gentianaceae, Primulaceae), habitats (species- rich grasslands), insect-pollinated and outcrossing species, and species mainly relying on sexual reproduction. On the other hand, few insights exist about grasses, ruderal plants and weeds, non-indigenous, wind- and self-pollinated species, and species mainly reproducing vegetatively or via apomictic seeds. However, according to the present state of knowledge especially these plant species, and those with a high dispersal potential, have to be considered as less sensitive to habitat fragmentation. Based on these findings, habitat types are classified with regard to their sensitivity to fragmentation, and ecological characters and species traits of sensitive and less sensitive species are compared. Finally, general consequences for conservation practice are presented with regard to target species and habitats for the formation of habitat networks, minimum viable population sizes, genetic rescue of populations, and deploying plants from ex-situ conservation to natural habitats.
We investigated the response of the microbial components of the pelagic food web to re-oligotrophication of large, deep Lake Constance where total phosphorus concentrations during mixing decreased from a maximum of 2.81 mu mol L- 1 in 1979 via 1.87 mu mol L-1 in 1987 to 0.26 mu mol L-1 in 2007. Measurements of heterotrophic bacteria, autotrophic picoplankton (APP) and heterotrophic nanoflagellates (HNF) in 2006 and 2007 were compared to values from 1987 to 1997. We hypothesized that the biomass and seasonal variability of all groups will decrease under more oligotrophic conditions due to reduced resource availability, particularly for APP and HNF but less for the competitively stronger bacteria. Average bacterial biomass between spring and autumn was unrelated to phosphorus, whereas the ratio of bacterial biomass to chlorophyll a concentration increased with decreasing trophy due to declining chlorophyll concentrations. In contrast, a unimodal relationship was found between APP and phosphorus with low biomass at low and high phosphorus concentrations and maximum biomass in between. Average HNF biomass decreased strongly by a factor of 10-30 with decreasing trophy, and chlorophyll-specific HNF biomass was unimodally related to phosphorus. The relative seasonal biomass variability did not change for any group during re-oligotrophication. To conclude, HNF responded much more strongly and bacteria less so than chlorophyll concentrations to oligotrophication, whereas APP exhibited a more complex pattern.
The zooplankton of oligotrophic lakes in North Patagonia is often dominated by mixotrophic ciliates, particularly Stentor amethystinus and Stentor araucanus. Therefore, we tested whether Stentor spp. (i) is an important food for juvenile endemic (Cheirodon australe, Galaxias maculatus, Odontesthes mauleanum, Percichthys trucha) and introduced (Oncorhynchus mykiss) fish species, and (ii) represents a remarkable grazer of bacteria. Ingestion rates of fish estimated by disappearance of Stentor in feeding experiments ranged between 8 (G. maculatus) and 53 (C australe) ciliates per fish and day, and assimilation rates measured by using radioactively labelled Stentor ranged between 3 (P. trucha) and 52 (C australe) ciliates per fish and day. However, although we detected the consumption of Stentor by fish, the daily consumption amounted to at most 0.2% of the fish biomass which can not cover the energy requirement of the fish. Furthermore, the daily consumption was equivalent to a maximum of 1.6% of the Stentor standing stock so that fish predation does not seem to be an important mortality factor for the ciliates. The clearance rate of Stentor sp. on natural bacteria was on average 3.8 mu l cil(-1) h(-1). The daily ingestion (mean 3.9 ngC cil(-1) d(-1)) was about 3.5% of the individual biomass of Stentor sp. Therefore, bacteria ingestion might explain a ciliate growth rate of appr. 1% d(-1), which was about 17% of the photosynthesis of endosymbiotic algae. The maximum density of Stentor sp. in the take could ingest about 1 mu g C L-1 d(-1) bacteria which is only 3% of average bacterial production. Thus, grazing by Stentor sp. does not seem to be a main loss factor for the bacteria.
Sequence variation of a fragment of the mitochondrial DNA encoding for the cytochrome b gene was used to reconstruct the phylogeography of the two species of bleaks occurring in Italy: the alborella Alburnus arborella in northern Italy and the vulturino Alburnus albidus in southern Italy. The study includes four populations of the alborella and 14 populations of the vulturino. A total of 57 haplotypes were identified; these could not be sorted into two reciprocally monophyletic clusters. Multiple phylogenetic methods and nested clade phylogeographical analysis consistently retrieved three well-supported clades, two of which contained both Northern and Southern Italian haplotypes. A third clade is limited to southern Italy. This clade is tentatively assigned to the vulturino. The placement in the same clade of northern and southern Italian haplotypes is explained in light of the introductions of fishes operated from northern to central and southern Italy. The origin of the vulturino dates back to the last two million years. This divergence time estimate identifies the Pleistocene confluences between adjacent river basins along the Adriatic slope of the Italian peninsula and their subsequent isolation as the cause that triggered the diversification of the genus in the area. The existence of a clade endemic to southern Italy supports the recognition of the area as a new peri-Mediterranean ichthyogeographic district, the borders of which correspond to the northern and southern edges of the vulturino range.
Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains
(2009)
Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well- characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other gamma-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3- mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.
Parallel A-helices are among the simplest repetitive structural elements in proteins. The folding behavior of A- helix proteins has been studied intensively, also to gain insight on the formation of amyloid fibrils, which share the parallel beta-helix as a central structural motif. An important system for investigating beta-helix folding is the tailspike protein from the Salmonella bacteriophage P22. The central domain of this protein is a right-handed parallel beta-helix with 13 windings. Extensive mutational analyses of the P22 tailspike protein have revealed two main phenotypes: temperature-sensitive-folding (tsf) mutations that reduce the folding efficiency at elevated temperatures, and global suppressor (su) mutations that increase the tailspike folding efficiency. A central question is whether these phenotypes can be understood from changes in the protein stability induced by the mutations. Experimental determination of the protein stability is complicated by the nearly irreversible trimerization of the folded tailspike protein. Here, we present calculations of stability changes with the program FoldX, focusing on a recently published extensive data set of 145 singe-residue alanine mutants. We find that the calculated stability changes are correlated with the experimentally measured in vivo folding efficiencies. In addition, we determine the free-energy landscape of the P22 tailspike protein in a nucleation-propagation model to explore the folding mechanism of this protein, and obtain a processive folding route on which the protein nucleates in the N-terminal region of the helix.
Life history traits within species often vary among different habitats. We measured female fecundity in mollies (Poecilia mexicana) from a H2S-rich cave and from a neighbouring surface habitat, as well as in laboratory-reared individuals of both populations raised in either light or continuous darkness. Compared to conspecifics from surface habitats, cave-dwelling P. mexicana had reduced fecundity (adjusted for size) in the field. In the laboratory, the fecundity of surface mollies was higher in light than in darkness, whereas fecundity in the cave mollies was almost unaffected by the ambient light conditions. Our results suggest a heritable component to the reduction in fecundity in female cave mollies. Moreover, the reduced plasticity in fecundity of cave mollies in response to light conditions might be an example of genetic assimilation or channelling of a life history trait in a population invading a new environment.
Crosses between plants from different populations may result in heterosis or outbreeding depression. However, despite its importance for conservation, little is known about the spatial scale over which these effects may arise. To investigate the consequences of between-population crosses at two distinct spatial scales, we conducted reciprocal crosses between four populations from two regions in the rare perennial herb Aster amellus. We assessed seed set and offspring fitness in a common garden experiment. Overall, between-population crosses within regions (10 km) resulted in 8% lower seed set than within-population crosses, while between-region crosses (70 km) resulted in 17% higher seed set than within-population crosses. Moreover, offspring from between-population crosses produced 18% more flower heads than offspring from within-population crosses. We conclude that hybridisation between A. amellus plants from different populations did not lead to immediate outbreeding depression and, thus, could represent a valid conservation option to increase genetic diversity. Moreover, our results suggest that the distance between populations affects the outputs of between-population crosses and therefore needs to be taken into account when promoting gene flow between populations.
The alpha-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant a-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of alpha-1,4-D-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. in this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.
Contrasting signals from multiple markers illuminate population connectivity in a marine fish
(2009)
Recent advances in molecular biology and bioinformatics have helped to unveil striking and previously unrecognized patterns of geographic genetic structure in marine populations. Largely driven by the pressing needs of fisheries management and conservation, studies on marine fish populations have played a pivotal role in testing the efficiency of a range of approaches to explore connectivity and dispersal at sea. Here, we employed nuclear and mitochondrial DNA markers and parasitic infestations to examine the nature and patterns of population structure in a warm-temperate coastal marine teleost across major putative biogeographic barriers in the Mediterranean Sea and Eastern Atlantic Ocean. We detected deep genetic divergence between mitochondrial lineages, likely caused by dramatic climatic and geological transformations before and during the Pleistocene. Such long-diverged lineages later came into secondary contact and can now be found in sympatry. More importantly, microsatellite data revealed that these lineages, after millions of years of independent evolution, now interbreed extensively. By combining genetic and parasite data, we were able to identify at least five independent demographic units. While the different genetic and parasite-based methods produce notably contrasting signals and may complicate the reconstruction of connectivity dynamics, we show that by tailoring the correct interpretation to each of the descriptors used, it is possible to achieve a deeper understanding of the micro-evolutionary process and, consequently, resolve population structure.
Similar to maternal care, paternal care is a source of neonatal sensory stimulation, which in primates and rodents has been shown to be essential for developing structure and function of sensory cortices. The aim of our study in the biparental rodent Octodon degus was to assess the impact of paternal deprivation on dendritic and synaptic development in the somatosensory cortex. We (i) quantified the amount of paternal care in relation to total parental investment and (ii) compared dendritic and synaptic development of pyramidal neurons in the somatosensory cortex of animals raised by a single mother or by both parents. On the behavioral level we show that paternal care comprises 37% of total parent-offspring interactions, and that the somatosensory stimulation provided by the fathers primarily consists of huddling, licking/grooming, and playing. On the morphological level we found that, compared with offspring raised by both parents (mother and father), the father-deprived animals displayed significantly reduced spine numbers on the basal dendrites of pyramidal neurons. Furthermore, paternal deprivation induces hemispheric asymmetry of the dendritic morphology of somatosensory pyramidal neurons. Father-deprived animals show shorter and less complex basal dendrites in the left somatosensory cortex compared with the right hemisphere. These findings indicate that paternal deprivation results in delayed or retarded dendritic and synaptic development of somatosensory circuits.
A test for conspecific cueing in two sympatric species of pupfish (Cyprinodon beltrani, C. simus)
(2009)
In many fishes, individuals prefer to associate with phenotypically similar or conspecific individuals (conspecific cueing). Such phenotypic segregation can be an important evolutionary driver, for example, in intralacustric sympatric speciation processes. I examined conspecific cueing in two species of sympatric pupfish from Laguna Chichancanab in southern Mexico: the little shoaling and highly territorial Cyprinodon beltrani and the highly shoaling but non-territorial C. simus. Females were tested for shoal species preferences in two testing scenarios: (1) a sequential choice test where shoals of four conspecific or four heterospecific (Cyprinodon sp. or Poecilia reticulata) females were presented in succession, and (2) a simultaneous choice test where female shoals of both Cyprindon species were presented concurrently. Overall, higher shoaling in C. simus was corroborated in this study. In the sequential test, no effect of the type of stimulus shoal (con- or heterospecific) on shoaling behavior was detected. In the simultaneous tests, C. beltrani, but not C. simus females showed a preference for the conspecific shoal. It seems possible that C. simus females did not evolve species recognition mechanisms because no other Cyprinodon species in the Laguna Chichancanab shows equally high shoaling, which automatically leads to the formation of single-species (i.e., C. simus-) shoals. C. simus males do not establish long-term territories, but rather spawn within shoals, whereas C. beltrani females approach males in their breeding territories to spawn. I discuss that this behavioral difference still provides a powerful reproductive isolation mechanism even in the absence of conspecific cueing in C. simus.
Aim To investigate the effect of temperature, latitude and local environment on the reproductive traits of widespread perennial forest herbs to better understand the potential impacts of rising temperatures on their population dynamics and colonization capacities. Location Six regions along a latitudinal gradient from France to Sweden. Methods Within each region, we collected data from three to five populations of up to six species. For each species, several variables were recorded in each region (temperature, latitude) and population (local abiotic and biotic environmental variables), and seed production and germination were estimated. Resource investment in reproduction (RIR) was quantified as seed number ¥ seed mass, while germinable seed output (GSO) was expressed as seed number ¥ germination percentage.We performed linear regression and mixed effect models to investigate the effects of temperature (growing degree hours), latitude and local abiotic and biotic environment on RIR and GSO. Results Temperature and latitude explained most of the variation in RIR and GSO for early flowering species with a northerly distribution range edge (Anemone nemorosa, Paris quadrifolia and Oxalis acetosella). Reproduction of the more southerly distributed species (Brachypodium sylvaticum, Circaea lutetiana and Primula elatior), in contrast, was independent of temperature/latitude. In the late summer species, B. sylvaticum and C. lutetiana, variation in RIR and GSO was best explained by local environmental variables, while none of the investigated variables appeared to be related to reproduction in P. elatior. Main conclusions We showed that reproduction of only two early flowering, northerly distributed species was related to temperature. This suggests that the potential reproductive response of forest herbs to climate warming partly depends on their phenology and distribution, but also that the response is to some extent species dependent. These findings should be taken into account when predictions about future shifts in distribution range are made.
Predictions on displacement of suitable habitats due to climate change suggest that plant species with poor colonization ability may be unable to move fast enough to match forecasted climate-induced changes in habitat distribution. However, studies on early Holocene plant migration show fast migration of many plant species that are poor colonizers today. We hypothesize that warmer temperatures during the early Holocene yielded higher seed quality, contributing to explaining the fast migration. We studied how the 3 seed quality variables, seed mass, germinability, and requirements for break of seed dormancy, vary for seeds of 11 forest herb species with varying colonization capacity collected along a 1400-km latitudinal gradient. Within species, seed mass showed a positive correlation with latitude, whereas germinability was more positively correlated with temperature (growing degree hours obtained at time of seed collection). Only slow-colonizing species increased germinability with temperature, whereas only fast-colonizing species increased germinability with latitude. These interactions were only detectable when analyzing germinability of the seeds, even though this trait and seed mass were correlated. The requirement for dormancy break did not correlate with latitude or temperature. The results indicate that seed development of slow colonizers may be favoured by a warmer climate, which in turn may be important for their migration capacity.
Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein Sufl can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.
Shrub encroachment linked to heavy grazing has dramatically changed savanna landscapes, and is a major form of rangeland degradation. Our understanding of how shrub encroachment affects arthropod communities is poor, however. Here, we investigate the effects of shrub encroachment on abundance and diversity of ground-dwelling (wingless) arthropods at varying levels of shrub cover in the southern Kalahari. We also ascertain if invertebrate assemblage composition changes with habitat structure and identify which aspects of habitat structure (e.g., grass cover, herbaceous plant cover, shrub density) correlate most strongly with these changes. Ant, scorpion and dung beetle abundance increased with shrub cover, whereas grasshoppers and solifuges declined. Spider and beetle abundance exhibited hump-shaped relationships with shrub cover. RTU richness within orders either mirrored abundances, or exhibited no trend. Shrub density was the habitat component most correlated with similarities between invertebrate assemblages. Ground-dwelling arthropods showed clear shifts in species assemblage composition at a similarity level of 65% according to shrub density. Changes in indicator species showed that within the Tenebrionidae (darkling beetles), certain species respond positively to shrub thickening, replacing other species within the Family. Small-bodied, wingless Scarabaeidae (dung beetles) tended to increase with increased shrub density and three species emerged as significant indicators of more thickened habitats, although this might be a response to greater dung availability, rather than habitat structure itself. We conclude that because ground- dwelling invertebrates showed such clear responses in species assemblage composition, they present excellent candidates for use as indicator species in further studies into bush encroachment.
The hypothesis that females of socially monogamous species obtain indirect benefits (good or compatible genes) from extra-pair mating behaviour has received enormous attention but much less generally accepted support. Here we ask whether selection for adult survival and fecundity or sexual selection contribute to indirect selection of the extra- pair mating behaviour in socially monogamous coal tits (Periparus ater). We tracked locally recruited individuals with known paternity status through their lives predicting that the extra-pair offspring (EPO) would outperform the within- pair offspring (WPO). No differences between the WPO and EPO recruits were detected in lifespan or age of first reproduction. However, the male WPO had a higher lifetime number of broods and higher lifetime number of social offspring compared with male EPO recruits, while no such differences were evident for female recruits. Male EPO recruits did not compensate for their lower social reproductive success by higher fertilization success within their social pair bonds. Thus, our results do not support the idea that enhanced adult survival, fecundity or within-pair fertilization success are manifestations of the genetic benefits of extra-pair matings. But we emphasize that a crucial fitness component, the extra-pair fertilization success of male recruits, has yet to be taken into account to fully appreciate the fitness consequences of extra-pair matings.
SDM performance varied for different range dynamics. Prediction accuracies decreased when abrupt range shifts occurred as species were outpaced by the rate of climate change, and increased again when a new equilibrium situation was realised. When ranges contracted, prediction accuracies increased as the absences were predicted well. Far- dispersing species were faster in tracking climate change, and were predicted more accurately by SDMs than short- dispersing species. BRTs mostly outperformed GLMs. The presence of a predator, and the inclusion of its incidence as an environmental predictor, made BRTs and GLMs perform similarly. Results are discussed in light of other studies dealing with effects of ecological traits and processes on SDM performance. Perspectives are given on further advancements of SDMs and for possible interfaces with more mechanistic approaches in order to improve predictions under environmental change.
Aim This study aims to link demographic traits and post-glacial recolonization processes with genetic traits in Himantoglossum hircinum (L.) Spreng (Orchidaceae), and to test the implications of the central-marginal concept (CMC) in Europe. Location Twenty sites covering the entire European distribution range of this species. Methods We employed amplified fragment length polymorphism (AFLP) markers and performed a plastid microsatellite survey to assess genetic variation in 20 populations of H. hircinum located along central-marginal gradients. We measured demographic traits to assess population fitness along geographical gradients and to test for correlations between demographic traits and genetic diversity. We used genetic diversity indices and analyses of molecular variance (AMOVA) to test hypotheses of reduced genetic diversity and increased genetic differentiation and isolation from central to peripheral sites. We used Bayesian simulations to analyse genetic relationships among populations. Results Genetic diversity decreased significantly with increasing latitudinal and longitudinal distance from the distribution centre when excluding outlying populations. The AMOVA revealed significant genetic differentiation among populations (F-ST = 0.146) and an increase in genetic differentiation from the centre of the geographical range to the margins (except for the Atlantic group). Population fitness, expressed as the ratio N-R/N, decreased significantly with increasing latitudinal distance from the distribution centre. Flower production was lower in most eastern peripheral sites. The geographical distribution of microsatellite haplotypes suggests post-glacial range expansion along three major migratory pathways, as also supported by individual membership fractions in six ancestral genetic clusters (C1-C6). No correlations between genetic diversity (e.g. diversity indices, haplotype frequency) and population demographic traits were detected. Main conclusions Reduced genetic diversity and haplotype frequency in H. hircinum at marginal sites reflect historical range expansions. Spatial variation in demographic traits could not explain genetic diversity patterns. For those sites that did not fit into the CMC, the genetic pattern is probably masked by other factors directly affecting either demography or population genetic structure. These include post-glacial recolonization patterns and changes in habitat suitability due to climate change at the northern periphery. Our findings emphasize the importance of distinguishing historical effects from those caused by geographical variation in population demography of species when studying evolutionary and ecological processes at the range margins under global change.
Starch is an important plant product widely used as a nutrient, as a source of renewable energy, and for many technological applications. In plants, starch is the almost ubiquitous storage carbohydrate whereas most heterotrophic prokaryotes and eukaryotes rely on glycogen. Despite close similarities in basic chemical features, starch and glycogen differ in both structural and physicochemical properties. Glycogen is a hydrosoluble macromolecule with evenly distributed branching points. Starch exists as a water-insoluble particle having a defined (and evolutionary conserved) internal structure. The biochemistry of starch requires the co-operation of up to 40 distinct (iso)enzymes whilst approximately 10 (iso)enzymes permit glycogen metabolism. The biosynthesis and degradation of native starch include the transition of carbohydrates from the soluble to the solid phase and vice versa. In this review, two novel aspects of the eukaryotic plastidial starch degradation are discussed: Firstly, biochemical reactions that take place at the surface of particulate glucans and mediate the phase transition of carbohydrates. Secondly, processes that occur downstream of the export of starch-derived sugars into the cytosol. Degradation of transitory starch mainly results in the formation of neutral sugars, such as glucose and maltose, that are transported into the cytosol via the respective translocators. The cytosolic metabolism of the neutral sugars includes the action of a hexokinase, a phosphoglucomutase, and a transglucosidase that utilizes high molecular weight glycans as a transient glucosyl acceptor or donor. Data are included on the transglucosidase (disproportionating isozyme 2) in Cyanophora paradoxa that accumulates storage carbohydrates in the cytosol rather than in the plastid.
In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of mono-phosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.
Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear alpha-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases alpha-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.
Of the four chloroplast beta-amylase (BAM) proteins identified in Arabidopsis, BAM3 and BAM4 were previously shown to play the major roles in leaf starch breakdown, although BAM4 apparently lacks key active site residues and beta- amylase activity. Here we tested multiple BAM4 proteins with different N-terminal sequences with a range of glucan substrates and assay methods, but detected no alpha-1,4-glucan hydrolase activity. BAM4 did not affect BAM1, BAM2 or BAM3 activity even when added in 10-fold excess, nor the BAM3-catalysed release of maltose from isolated starch granules in the presence of glucan water dikinase. However, BAM4 binds to amylopectin and to amylose-Sepharose whereas BAM2 has very low beta-amylase activity and poor glucan binding. The low activity of BAM2 may be explained by poor glucan binding but absence of BAM4 activity is not. These results suggest that BAM4 facilitates starch breakdown by a mechanism involving direct interaction with starch or other alpha-1,4-glucan.
In plants several 'starch-related' enzymes exist as plastid- and cytosol-specific isoforms and in some cases the extraplastidial isoforms represent the majority of the enzyme activity. Due to the compartmentation of the plant cells, these extraplastidial isozymes have no access to the plastidial starch granules and, therefore, their in vivo function remained enigmatic. Recently, cytosolic heteroglycans have been identified that possess a complex pattern of the monomer composition and glycosidic bonds. The glycans act both as acceptors and donors for cytosolic glucosyl transferases. In autotrophic tissues the heteroglycans are essential for the nocturnal starch-sucrose conversion. In this review we summarize the current knowledge of these glycans, their interaction with glucosyl transferases and their possible cellular functions. We include data on the heteroglycans in heterotrophic plant tissues and discuss their role in intracellular carbon fluxes that originate from externally supplied carbohydrates.
The main objective of this study was to identify genomic regions involved in biomass heterosis using QTL, generation means, and mode-of-inheritance classification analyses. In a modified North Carolina Design III we backcrossed 429 recombinant inbred line and 140 introgression line populations to the two parental accessions, C24 and Col-0, whose F 1 hybrid exhibited 44% heterosis for biomass. Mid-parent heterosis in the RILs ranged from −31 to 99% for dry weight and from −58 to 143% for leaf area. We detected ten genomic positions involved in biomass heterosis at an early developmental stage, individually explaining between 2.4 and 15.7% of the phenotypic variation. While overdominant gene action was prevalent in heterotic QTL, our results suggest that a combination of dominance, overdominance and epistasis is involved in biomass heterosis in this Arabidopsis cross.
Carbon assimilation mode in mixotrophs and the fatty acid composition of their rotifer consumers
(2009)
P>1. We examined an important ecophysiological link between the mixotrophic flagellate Chlamydomonas acidophila and its consumers, the rotifers Elosa worallii, Cephalodella sp. and Brachionus sericus, by comparing their fatty acid profiles. 2. The mixotrophic flagellate was grown under either exclusively autotrophic conditions in the light, under exclusively heterotrophic conditions in the dark with an organic carbon source (glucose), or in the light plus the organic carbon sources (=mixotrophic). 3. Under heterotrophic growth conditions, C. acidophila strongly reduced its content of the n-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALA, C18:3n-3) compared with auto- and mixotrophic growth conditions. Although PUFAs with more than 18 carbon atoms were not detected in C. acidophila, significant amounts of eicosatetraenoic (ETA, 20:4n-3) and eicosapentaenoic acid (EPA, 20:5n-3) were found in three rotifer consumers. 4. Species-specific differences in the fatty acid profiles with respect to ETA, EPA and the precursor ALA were found in the rotifers: Brachionus and Cephalodella fed on the heterotrophic diets synthesised less EPA. In Elosa, smaller amounts of ALA were detected but were converted efficiently to a constant content of EPA and to an exceptionally high content of ETA. 5. Since in nature the mode of carbon assimilation among mixotrophic organisms differs, and their fatty acid composition varies depending on their mode of carbon assimilation, the availability of ALA might be critical for their consumers. An insufficient dietary supply of this precursor for the synthesis of ETA and EPA can prevent consumers from regulating their content of ETA and EPA. Therefore, observed differences in values of the latter might underly species-specific differences in the competitive capability of consumers.
Enhanced water temperatures promote the occurrence of cyanobacterial blooms, which may be detrimental to aquatic herbivores. Especially, the often-dominant crustaceans could be negatively affected because cyanobacteria are deficient in phytosterols, which are required by the crustaceans to form the membrane component cholesterol, which in turn plays a role in thermal adaptation. Here, we determined the influence of temperature on growth, reproduction and the allocation of dietary sterol into somatic tissues and eggs of the keystone species Daphnia magna raised along a dietary cholesterol gradient. Mass-specific growth rates of D. magna increased with the increasing availability of dietary cholesterol up to an incipient limiting level, which increased with increasing temperature. This indicates a higher demand for cholesterol for growth at higher temperatures and may explain the consistently smaller clutch sizes of reproducing females at the highest temperature. The cholesterol content of the individuals increased with increasing dietary cholesterol; this increase was enhanced at higher temperatures, indicating a higher demand for cholesterol for tissues and probably specifically for membranes. Surprisingly, the daphnids showed different allocation strategies with regard to temperature and dietary sterol availability. The cholesterol content of eggs was enhanced at higher temperature, which suggested that females allocate more cholesterol to their offspring, presumably to ensure sufficient egg development. When dietary cholesterol was limiting, however, females did not allocate more cholesterol to their eggs. Our data suggest that during cyanobacterial blooms, a potential dietary sterol limitation of Daphnia can be intensified at higher water temperatures, which can occur with global warming.
Prediction of hybrid biomass in Arabidopsis thaliana by selected parental SNP and metabolic markers
(2009)
A recombinant inbred line (RIL) population, derived from two Arabidopsis thaliana accessions, and the corresponding testcrosses with these two original accessions were used for the development and validation of machine learning models to predict the biomass of hybrids. Genetic and metabolic information of the RILs served as predictors. Feature selection reduced the number of variables (genetic and metabolic markers) in the models by more than 80% without impairing the predictive power. Thus, potential biomarkers have been revealed. Metabolites were shown to bear information on inherited macroscopic phenotypes. This proof of concept could be interesting for breeders. The example population exhibits substantial mid-parent biomass heterosis. The results of feature selection could therefore be used to shed light on the origin of heterosis. In this respect, mainly dominance effects were detected.
Background: The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent Onchip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. Methods: HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the Onchip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. Results: We have successfully shown the OnChip-HDA and applied it for single- and duplex- detection of the pathogens N. gonorrhoeae and S. aureus. Conclusion: We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.
Motivation: Full-length DNA and protein sequences that span the entire length of a gene are ideally used for multiple sequence alignments (MSAs) and the subsequent inference of their relationships. Frequently, however, MSAs contain a substantial amount of missing data. For example, expressed sequence tags (ESTs), which are partial sequences of expressed genes, are the predominant source of sequence data for many organisms. The patterns of missing data typical for EST-derived alignments greatly compromise the accuracy of estimated phylogenies. Results: We present a statistical method for inferring phylogenetic trees from EST-based incomplete MSA data. We propose a class of hierarchical models for modeling pairwise distances between the sequences, and develop a fully Bayesian approach for estimation of the model parameters. Once the distance matrix is estimated, the phylogenetic tree may be constructed by applying neighbor-joining (or any other algorithm of choice). We also show that maximizing the marginal likelihood from the Bayesian approach yields similar results to a pro. le likelihood estimation. The proposed methods are illustrated using simulated protein families, for which the true phylogeny is known, and one real protein family.
Empirical data providing evidence for a colimitation of an herbivore by two or more essential nutrients are scarce, particularly in regard to biochemical resources. Here, a graphical model is presented, which describes the growth of an herbivore in a system with two potentially limiting resources. To verify this model, life-history experiments were conducted with the herbivore Daphnia magna feeding on the picocyanobacterium Synechococcus elongatus, which was supplemented with increasing amounts of cholesterol either in the presence or the absence of saturating amounts of eicosapentaenoic acid (EPA). For comparison, D. magna was raised on diets containing different proportions of S. elongatus and the cholesterol- and EPA-rich eukaryotic alga Nannochloropsis limnetica. Somatic and population growth of D. magna on a sterol- and EPA-deficient diet was initially constrained by the absence of sterols. With increased sterol availability, a colimitation by EPA became apparent and when the sterol requirements were met, the growth- limiting factor was shifted from a limitation by sterols to a limitation by EPA. These data imply that herbivores are frequently limited by two or more essential nutrients simultaneously. Hence, the concept of colimitation has to be incorporated into models assessing nutrient-limited growth kinetics of herbivores to accurately predict demographic changes and population dynamics.
Enzymatic isothermal rolling circle amplification (RCA) produces long concatemeric single-stranded DNA (ssDNA) molecules if a small circular ssDNA molecule is applied as the template. A method is presented here in which the RCA reaction is carried out in a flow-through system, starting from isolated surface-tethered DNA primers. This approach combines gentle fluidic handling of the single-stranded RCA products, such as staining or stretching via a receding meniscus, with the option of simultaneous (fluorescence) microscopic observation. It is shown that the stretched and surface-attached RCA products are accessible for hybridization of complementary oligonucleotides, which demonstrates their addressability by complementary base pairing. The long RCA products should be well suited to bridge the gap between biomolecular nanoscale building-blocks and structures at the micro- and macroscale, especially at the single- molecule level presented here.
Isothermal amplification technologies are emerging on the horizon that could have the potential to pose as alternatives to PCR in terms of sensitivity and ease of use. One of the most recent isothermal technologies is helicase- dependent amplification (HDA). This technology uses the helicase's capability to disrupt the hydrogen bonds of a Watson-Crick base pair in order to separate dsDNA. A denaturation step, as is used in PCR, is no longer required. This gives rise to new, less expensive and less complicated designs for point-of-care devices and 'Lab on Chip' systems. Helicase-dependent OnChip-amplification (OnChip-HDA) is a further step into this direction as it integrates the HDA technology with microarray technology and its power of multiplexing. This special report will give an overview on the HDA and OnChip-HDA technology, and its potential for point-of-care diagnostics.
This work describes a cell-based assay that does not depend on radioactivity or laboratory animals for the detection of ligands of angiotensin II type 1 receptor (AT(1)R). The assay makes use of stable transfected Chinese hamster ovary cells (CHO-AT(1)R) expressing the AT(1)R. A sequential saturation assay principle was used in which receptor binding sites of the CHO-AT(1)R cells are blocked by the analyte in a concentration-dependent manner. Afterwards, TAMRA-angiotensin II, a fluorescence-labeled ligand, was added to bind to the remaining free binding sites of the receptor. In consequence, the fluorescence signal determined is inversely proportional to the concentration of the analyte.