Ultrasound radiation force modulates ligand availability on targeted contrast agents

Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.     

Monoacylglycerol Lipases Act as Evolutionarily Conserved Regulators of Non-oxidative Ethanol Metabolism

Fatty acid ethyl esters (FAEEs) are non-oxidative metabolites of ethanol that accumulate in human tissues upon ethanol intake. Although FAEEs are considered as toxic metabolites causing cellular dysfunction and tissue damage, the enzymology of FAEE metabolism remains poorly understood. In this study, we used a biochemical screen in Saccharomyces cerevisiae to identify and characterize putative hydrolases involved in FAEE catabolism. We found that Yju3p, the functional orthologue of mammalian monoacylglycerol lipase (MGL), contributes >90% of cellular FAEE hydrolase activity, and its loss leads to the accumulation of FAEE. Heterologous expression of mammalian MGL in yju3Δ mutants restored cellular FAEE hydrolase activity and FAEE catabolism. Moreover, overexpression or pharmacological inhibition of MGL in mouse AML-12 hepatocytes decreased or increased FAEE levels, respectively. FAEEs were transiently incorporated into lipid droplets (LDs) and both Yju3p and MGL co-localized with these organelles. We conclude that the storage of FAEE in inert LDs and their mobilization by LD-resident FAEE hydrolases facilitate a controlled metabolism of these potentially toxic lipid metabolites.     

Clone-specific responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress

The effects of enhanced UVB radiation and drought stress on willow secondary phenolics were studied using the leaves of 8‐week‐old micropropagated plantlets from interspecific hybrids (Salix myrsinites L. ×S. myrsinifolia Salisb.) and pure species (S. myrsinifolia). The plantlets were subjected for 4 weeks to two levels of UVB radiation (ambient, enhanced) and two levels of watering (well‐watered, drought‐stressed) according to a 2 × 2 factorial design. Enhanced UVB radiation increased the total concentration of flavonoids and phenolic acids in all plantlets, while the total concentration of salicylates remained unaffected. Drought stress reduced the total concentration of salicylates and phenolic acids in S. myrsinifolia plantlets, while in hybrids only phenolic acids were affected. The response of phenolic acids to enhanced UVB in drought‐stressed plantlets was different from that in well‐watered ones, indicating that drought stress limited the accumulation of phenolic acids under enhanced UVB radiation. Flavonoids increased in response to enhanced UVB radiation in drought‐stressed plantlets, although drought caused serious physiological stress on growth. There were significant differences between hybrid and S. myrsinifolia plantlets with respect to the composition of phenolics and between families and clones with respect to their concentration. In addition, the response of salicylates, flavonoids and phenolic acids to enhanced UVB and drought stress was clone‐specific, which may indicate that climatic changes will alter the genetic composition of northern forests.     

Long-distance migration: evolution and determinants

Long‐distance migration has evolved in many organisms moving through different media and using various modes of locomotion and transport. Migration continues to evolve or become suppressed as shown by ongoing dynamic and rapid changes of migration patterns. This great evolutionary flexibility may seem surprising for such a complex attribute as migration. Even if migration in most cases has evolved basically as a strategy to maximise fitness in a seasonal environment, its occurrence and extent depend on a multitude of factors. We give a brief overview of different factors (e.g. physical, geographical, historical, ecological) likely to facilitate and/or constrain the evolution of long‐distance migration and discuss how they are likely to affect migration. The basic driving forces for migration are ecological and biogeographic factors like seasonality, spatiotemporal distributions of resources, habitats, predation and competition. The benefit of increased resource availability will be balanced by costs associated with the migratory process in terms of time (incl. losses of prior occupancy advantages), energy and mortality (incl. increased exposure to parasites). Furthermore, migration requires genetic instructions (allowing substantial room for learning in some of the traits) about timing, duration and distance of migration as well as about behavioural and physiological adaptations (fuelling, organ flexibility, locomotion, use of environmental transport etc) and control of orientation and navigation. To what degree these costs and requirements put constraints on migration often depends on body size according to different scaling relationships. From this exposé it is clear that research on migration warrants a multitude of techniques and approaches for a complete as possible understanding of a very complex evolutionary syndrome. In addition, we also present examples of migratory distances in a variety of taxons. In recent years new techniques, especially satellite radio telemetry, provide new information of unprecedented accuracy about journeys of individual animals, allowing re‐evaluation of migration, locomotion and navigation theories.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.     

Microbial Colonization of Beech and Spruce Litter—Influence of Decomposition Site and Plant Litter Species on the Diversity of Microbial Community

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the A_h horizon of beech and beech–spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT–PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT–PCR products from both litter types incubated in beech–spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by γ-proteobacterial members, whereas spruce libraries were mainly composed of α-, β-, and γ-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.