Host islands within the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon

We have examined fine-scale genetic structure of the symbiotic ectomycorrhizal fungi Rhizopogon occidentalis and R. vulgaris on two of the California Channel Islands using five and six microsatellite loci, respectively. Both Rhizopogon species are sympatric on Santa Cruz and Santa Rosa Islands and are ectomycorrhizal with bishop pine (Pinus muricata) on both islands or Santa Rosa Island Torrey pine (P. torreyana ssp. insularis) on Santa Rosa. The combination of disjunct pine host distributions and geographic barriers within and among the islands have created highly structured Rhizopogon populations over very short distances (8.5 km on Santa Cruz Island; F(ST) = 0.258, F(ST) = 0.056, R. occidentalis and R. vulgaris, respectively). Both species show similar patterns of genetic differentiation as a result of limited dispersal between host populations as revealed by a significant isolation by distance relationship (r = 0.69, P < 0.04; r = 0.93, P < 0.001, R. occidentalis and R. vulgaris, respectively) and Bayesian clustering analyses, and is most likely a function of the small foraging range of the few mammals that disperse Rhizopogon on these islands and the enormous spore bank characteristic of Rhizopogon species.     

A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees

AIMS: The goal of this research was the development of a PCR-based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. METHODS AND RESULTS: Eleven taxon-specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus/Phellinus-group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. CONCLUSIONS: The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.     

Persistent Effectivity of Gas Plasma-Treated,Long Time-Stored Liquid on Epithelial Cell Adhesion Capacity and Membrane Morphology

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco''s Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.     

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes,melanocytes and fibroblasts

Background  

The Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.     

Marsupial light chains: IGK with four V families in the opossum Monodelphis domestica

 A full-length and several partial cDNAs encoding IGK light chains from the marsupial South American opossum, Monodelphis domestica, were isolated and characterized. Using these clones as a starting point, the expressed IGKV repertoire was sampled by anchored polymerase chain reaction using an IGKC-specific primer. Based on nucleotide sequences of twenty unique, expressed IGKV-J combinations, there are at least four IGKV families and two J segments. Southern blot analysis revealed each IGK-V family contains multiple gene segments totaling at least thirty-five IGKV in the opossum genome. No evidence for particular, recurrent IGKV-J combinations in the opossum IGK repertoire was seen, rather the V-J combinations appeared random and diverse. Each of the four IGKV families appear more closely related to V segments from placental mammals than to each other, suggesting the duplication of the IGKV families prior to the separation of marsupials and placental mammals more than one-hundred-million years ago. Overall, the complexity of opossum light chain V segments appears greater than that found in the heavy chain, and light chains are likely to contribute significantly to Ig diversity in this species.With this report, the homologues encoding all three classes of eutherian Ig chains, IGH, IGL, and IGK, have been described in a non-placental mammal. Received: 5 April 1999 / Revised: 3 June 1999     

Quantification of specific bindings of biomolecules by magnetorelaxometry

The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP), to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX). Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated) serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.