Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.     

Land use and host neighbor identity effects on arbuscular mycorrhizal fungal community composition in focal plant rhizosphere

Arbuscular mycorrhizal fungi (AMF) provide a number of ecosystem services as important members of the soil microbial community. Increasing evidence suggests AMF diversity is at least partially controlled by the identities of plants in the host plant neighborhood. However, much of this evidence comes from greenhouse studies or work in invaded systems dominated by single plant species, and has not been tested in species-rich grasslands. We worked in 67 grasslands spread across the three German Biodiversity Exploratories that are managed primarily as pastures and meadows, and collected data on AMF colonization, AMF richness, AMF community composition, plant diversity, and land use around focal Plantago lanceolata plants. We analyzed the data collected within each Exploratory (ALB Schwäbische Alb, HAI Hainich-Dün, SCH Schorfheide-Chorin) separately, and used variance partitioning to quantify the contribution of land use, host plant neighborhood, and spatial arrangement to the effect on AMF community composition. We performed canonical correspondence analysis to quantify the effect of each factor independently by removing the variation explained by the other factors. AMF colonization declined with increasing land use intensity (LUI) along with concurrent increases in non-AMF, suggesting that the ability of AMF to provide protection from pathogens declined under high LUI. In ALB and HAI mowing frequency and percent cover of additional P. lanceolata in the host plant neighborhood were important for AMF community composition. The similar proportional contribution of land use and host neighborhood to AMF community composition in a focal plant rhizosphere suggests that the diversity of this important group of soil microbes is similarly sensitive to changes at large and small scales.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.     

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed. Received: 19 November 1998 / Accepted: 4 March 1999     

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.     

Complete elimination of hostplant quinolizidine alkaloids by larvae of a polyphagous lycaenid butterfly,Callophrys rubi

Caterpillars of the lycaenid butterfly Callophrys rubi accept a variety of hostplants. When fed inflorescences or leaves of Genista tinctoria (a natural hostplant) or Lupinus polyphyllus (a non-host), the larvae completely eliminate quinolizidine alkaloids ingested from their food in their frass. No alkaloids are stored. Infestation by the parasitoid wasp Distatrix sancus (Braconidae) did not affect alkaloid elimination. The presence of an effective anti-toxin system is discussed with reference to the evolution of hostplant relationships in the genus Callophrys. There is no evidence that in the secondarily myrmecoxenous larvae of C. rubi hostplantderived chemical defense takes the place of former myrmecophily.     

A cyp19a1b‐gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells

Aromatase is an enzyme that catalyzes the synthesis of estrogen in gonads and brain. Teleost fish express aromatase (AroB) strongly in the brain facilitating its detailed examination. To understand the function of AroB in the brain, we generated transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the brain aromatase cyp19a1b promoter. GFP was found in the radial glial cells of transgenic larvae and adult fish that overlap with AroB immunoreactivity in the correct temporal and spatial pattern. GFP was also coexpressed with radial cell marker BLBP, but was not in neurons. In addition, GFP expression in the radial glial cells was stimulated by estrogen, same as endogenous AroB expression. Thus, this transgenic line faithfully mimics the regulation of AroB expression in radial glial cells. It provides a powerful tool to further characterize progenitor radial cells in adult and developing fish and to evaluate estrogenic activities of xenoestrogens and phytoestrogens. genesis 47:67–73, 2009. © 2008 Wiley‐Liss, Inc.     

Halimeda contribution to organic and inorganic production in a Tahitian reef system

Of the seven species of Halimeda inhabiting a lagoon on Moorea island, three representing 10% of the algal covering and averaging 111 g of dry weight m^-2, have been studied in the course of a year. The biomass, measured bimonthly, stresses a slight seasonal variability in the species life. The main decrease was reported for H. opuntia after a fruiting event, which happened in October. The primary production was assessed, in situ, periodically over a year, by measuring oxygen variations in enclosures. Either expressed on specific-weight basis or in area units, the highest primary productions were recorded for H. opuntia. Productions and biomasses vary simultaneously during the year. The three species produce all together about 6 g C m^-2y^-1. The growth rate of the sand-dwelling H. incrassata f. ovata was followed during the year by staining, in the field, individuals with alizarin Red-S. The average rates measured were 3.3 segments ind^-1d^-1 and 0.17 gdw d^-1m^-2. The contribution of the three species to the carbonate budget of the reef was estimated by total alkalinity measurements during 24-h cycles. H. opuntia had the highest CaCO₃ production. For the three species studied, CaCO₃ production of 1.4 kg m^-2y^-1, which could correspond to a 0.4mm/year accretion of the studied reefal system, was estimated.     

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria

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.