Non-invasive LC-PolScope imaging of biominerals and cell wall anisotropy changes

The formation of defined shapes by cells is one of the challenging questions in biology. Due to the anisotropy of cell walls and of certain biominerals, the LC-PolScope represents a promising tool for tracking dynamic structural changes in vivo non-invasively and, to some extent, quantitatively. A complex three-dimensional biogenic system, the in vitro precipitation of calcium oxalate induced by cellulose stalks produced by Dictyostelium discoideum, was analyzed. Although the retardance values and orientation of the crystals with respect to the stalk were quickly and easily detected, this study raised a number of issues that were addressed in this work. The effect of the refractive index of the embedding medium was examined by taking advantage of the homogeneous size and shape distribution of kiwifruit raphides, a biologically controlled calcium oxalate biomineral and of cotton (Gossypium) seed fibers. The retardance remained consistent when embedding these samples in media with increasing refractive indices from 1.33 to 1.42 or 1.47 for sucrose or glycerol gradients, respectively. The general applicability of LC-PolScope image processing for biominerals and cell wall formation during development in vivo was demonstrated in a particular group of green algae, the Desmidiaceae. Various organization levels of the cell wall were identified, thus confirming earlier findings based on electron microscopy and immunostaining investigations. It can be concluded that LC-PolScope microscopy is an attractive tool for studying dynamic ordering of biomolecules, such as plant cell walls, when additional parameters regarding the structure, composition, and refractive indices of the specimen are available.     

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schr?der, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., H?llt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.     

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.     

Dissecting Arabidopsis Gβ signal transduction on the protein surface

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits.     

Memory in Elementary School Children Is Improved by an Unrelated Novel Experience

Education is the most traditional means with formative effect on the human mind, learning and memory being its fundamental support. For this reason, it is essential to find different strategies to improve the studentś performance. Based on previous work, we hypothesized that a novel experience could exert an enhancing effect on learning and memory within the school environment. Here we show that novel experience improved the memory of literary or graphical activities when it is close to these learning sessions. We found memory improvements in groups of students who had experienced a novel science lesson 1 hour before or after the reading of a story, but not when these events were 4 hours apart. Such promoting effect on long-term memory (LTM) was also reproduced with another type of novelty (a music lesson) and also after another type of learning task (a visual memory). Interestingly, when the lesson was familiar, it failed to enhance the memory of the other task. Our results show that educationally relevant novel events experienced during normal school hours can improve LTM for tasks/activities learned during regular school lessons. This effect is restricted to a critical time window around learning and is particularly dependent on the novel nature of the associated experience. These findings provide a tool that could be easily transferred to the classroom by the incorporation of educationally novel events in the school schedule as an extrinsic adjuvant of other information acquired some time before or after it. This approach could be a helpful tool for the consolidation of certain types of topics that generally demand a great effort from the children.     

Chiral bias of amyloid fibrils revealed by the twisted conformation of Thioflavin T: an induced circular dichroism/DFT study

Since it was implicated in a number of neurodegenerative conditions, such as Alzheimer disease, formation of beta-sheet-rich protein fibrils (amyloids) has been drawing a lot of attention. One of elusive aspects of amyloidogenesis concerns the mechanisms of specific binding of molecules such as Congo red, or Thioflavin T by amyloid fibrils. A comprehensive understanding of these docking interactions is needed, however, for the sake of furthering biochemical studies and developing molecular, pharmacological strategies preventing proliferation of amyloids in vivo. Through the application of circular dichroism, here we show that upon binding to insulin fibrils, a twisted conformation is enforced in molecules of Thioflavin T, manifested in a strong negative Cotton effect around 450 nm, which is supported by density functional theory-based calculations. This finding may lead to circular dichroism of Thioflavin T becoming a new diagnostic technique for protein fibrils, complementary to fluorescence spectroscopy.     

Breoghania corrubedonensis gen. nov. sp. nov., a novel alphaproteobacterium isolated from a Galician beach (NW Spain) after the Prestige fuel oil spill,and emended description of the family Cohaesibacteraceae and the species Cohaesibacter gelatinilyticus

A Gram-negative bacterium designated UBF-P1^T was isolated from an enrichment culture established in nutrient supplemented artificial sea water with pyrene as a carbon source, and inoculated with a marine fuel oil-degrading consortium obtained from a sand sample collected from the beach of Corrubedo (A Coruña, Galicia, Spain) after the Prestige accidental oil spill. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence affiliated strain UBF-P1^T with the family Cohaesibacteraceae, Cohaesibacter gelatinilyticus (DSM 18289^T) being the closest relative species with 92% sequence similarity. Cells were irregular rods, motile, strictly aerobic, catalase and oxidase positive. Ubiquinone 10 was the major respiratory lipoquinone. The major polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), and phosphatidylcholine (PC). The major fatty acids detected were C_18:1ω7c, C_19:0 cycloω8c, and C_16:0. The G + C content of strain UBF-P1^T was 63.9 mol%. The taxonomic comparison with the closest relative based on genotypic, phenotypic and chemotaxonomic characteristics supported that strain UBF-P1^T could be classified as a novel genus and species, for which the name Breoghania corrubedonensis gen. nov., sp. nov. is proposed. The type strain of this new taxon is UBF-P1^T (CECT 7622, LMG 25482, DSM 23382).