Separation of plant membranes by electromigration techniques

The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.     

Plant phylogeny drives arboreal caterpillar assemblages across the Holarctic

1. Assemblages of insect herbivores are structured by plant traits such as nutrient content, secondary metabolites, physical traits, and phenology. Many of these traits are phylogenetically conserved, implying a decrease in trait similarity with increasing phylogenetic distance of the host plant taxa. Thus, a metric of phylogenetic distances and relationships can be considered a proxy for phylogenetically conserved plant traits and used to predict variation in herbivorous insect assemblages among co‐occurring plant species.
2. Using a Holarctic dataset of exposed‐feeding and shelter‐building caterpillars, we aimed at showing how phylogenetic relationships among host plants explain compositional changes and characteristics of herbivore assemblages.
3. Our plant–caterpillar network data derived from plot‐based samplings at three different continents included >28,000 individual caterpillar–plant interactions. We tested whether increasing phylogenetic distance of the host plants leads to a decrease in caterpillar assemblage overlap. We further investigated to what degree phylogenetic isolation of a host tree species within the local community explains abundance, density, richness, and mean specialization of its associated caterpillar assemblage.
4. The overlap of caterpillar assemblages decreased with increasing phylogenetic distance among the host tree species. Phylogenetic isolation of a host plant within the local plant community was correlated with lower richness and mean specialization of the associated caterpillar assemblages. Phylogenetic isolation had no effect on caterpillar abundance or density. The effects of plant phylogeny were consistent across exposed‐feeding and shelter‐building caterpillars.
5. Our study reveals that distance metrics obtained from host plant phylogeny are useful predictors to explain compositional turnover among hosts and host‐specific variations in richness and mean specialization of associated insect herbivore assemblages in temperate broadleaf forests. As phylogenetic information of plant communities is becoming increasingly available, further large‐scale studies are needed to investigate to what degree plant phylogeny structures herbivore assemblages in other biomes and ecosystems.

     

Screening method for microorganisms producing L-aminoacids from D,L-5-monosubstituted hydantoins

Summary The ability of microorganisms to produce hydantoinase and L-N-carbamoylase could be established by an overlay assay. Enzyme producing strains form clear areas around their colonies caused by the cleavage of D,L-indolylmethylhydantoin. A second overlayer with a tryptophan-auxotroph yeast enables us to detect microorganisms which are able to produce L-tryptophan from D,L-indolylmethylhydantoin.     

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Diversification of Hawaiian Cyrtandra (Gesneriaceae) under the influence of incomplete lineage sorting and hybridization

Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 89 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian Cyrtandra lineage, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high‐throughput sequencing, and modern phylogenomics tools to test 31 Hawaiian Cyrtandra samples (22 species, two putative hybrids, four species with two samples each, one species with four samples) and two outgroups for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species‐tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. We expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and species‐tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Multiple hybridizations were inferred, but many were on deeper branches of the island lineages suggesting a long history of hybridization. We demonstrated the utility of high‐throughput sequencing and a phylogenomic approach using 569 loci to understanding species relationships and gene flow in the presence of ILS.     

Quantitative histology of the hypertrophied human heart

Myocardial hypertrophy accompanies systemic hypertension and aortic stenosis, i.e., pressure overload. In man cardiac failure only appears after years of pressure overload, during which time cardiac function had been maintained. The structural correlates of cardiac failure have been a subject of much interest for many years. Several hypotheses relating alterations in muscle fiber alignment, capillary density, or collagen content have been offered. The application of morphometric techniques has provided essential quantitative information on the structural components of the normal and diseased heart. These data indicate that muscle fiber alignment remains normal in the pressure overloaded heart despite the presence of hypertrophy or the appearance of clinical failure. On the other hand, capillary density is decreased and collagen content is increased in hypertrophied hearts. Chemical studies on collagen concentration however have yielded inconsistent results. The relative contribution of the microcirculation and collagenous structure of the myocardium on its respective O2 availability, mechanical behavior, and deterioration in pump function will require further investigation.     

Protection of Mice against Lethal Coxsackievirus B3 Infection by Using DNA Immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.