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101.
Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera 总被引:1,自引:0,他引:1
Kyselková M Kopecký J Felföldi T Cermák L Omelka M Grundmann GL Moënne-Loccoz Y Ságová-Marecková M 《Antonie van Leeuwenhoek》2008,94(3):439-453
Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions. 相似文献
102.
Maxime Willems Mieke Boone Marjolein Couvreur Katrien De Mulder Jelka Van Ranst Tom Artois Gaëtan Borgonie 《Development genes and evolution》2009,219(5):273-279
A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful
immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles
or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than
classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated
by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
103.
104.
Barthélemy X Bouvier G Radunz A Docquier S Schmid GH Franck F 《Photosynthesis research》2000,64(1):63-76
The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within
(etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein
contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using
a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide
in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased
in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes
at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes
of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid
regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This
suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
105.
Questions: How does disturbance and successional age influence richness, size and composition of the soil seed bank? What is the potential contribution of the soil seed bank to the plant community composition on sites differing in their successional age or disturbance intensity? Location: Experimental Botanical Garden of Göttingen University, central Germany. Methods: Above‐ground vegetation and soil seed bank were studied on formerly arable fields in a 36‐year‐old permanent plot study with five disturbance intensities, ranging from yearly ploughing via mowing to long‐term uninterrupted succession. We compared species compositions, seed densities and functional features of the seed bank and above‐ground vegetation by using several methods in parallel. Results: The seed bank was mainly composed of early successional species typical of strongly disturbed habitats. The difference between seed bank composition and above‐ground vegetation decreased with increasing disturbance intensity. The species of greatest quantitative importance in the seed bank was the non‐native forb Solidago canadensis. Conclusions: The ability of a plant community to regenerate from the soil seed bank dramatically decreases with increasing time since abandonment (successional age) and with decreasing disturbance intensity. The present study underlines that plant species typical of grasslands and woodlands are limited by dispersal capacity, owing to low capacity for accumulation of seeds in the soil and the fact that most species do not build up persistent seed banks. Rare and target species were almost absent from the seed bank and will, after local elimination, depend on reintroduction for continuation of their presence. 相似文献
106.
Background
Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.Methodology/Principal Findings
In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.Conclusion
The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies. 相似文献107.
Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria 总被引:1,自引:0,他引:1
Sanguin H Herrera A Oger-Desfeux C Dechesne A Simonet P Navarro E Vogel TM Moënne-Loccoz Y Nesme X Grundmann GL 《Environmental microbiology》2006,8(2):289-307
The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems. 相似文献
108.
ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate. 相似文献
109.
110.
Élodie Breton Christian Goetz Jacqueline Kintz Nathalie Accart Gaëlle Aubertin Bernard Grellier Philippe Erbs Ronald Rooke André Constantinesco Philippe Choquet 《Comptes rendus biologies》2010,333(3):220-225