Glutamate receptors and the airways hyperreactivity

It is proposed the link between the hyperactivity of NMDA receptors and airway hyperresponsiveness. We investigated the effect of agents modulating the activity of NMDA receptors in the ovalbumin-induced airway hyperreactivity in guinea pigs. The airways hyperreactivity was influenced by the agonist (NMDA) and selective antagonist - competitive (AP-5) and non-competitive (MK-801) of NMDA receptors. Airway responsiveness to histamine or acetylcholine was evaluated in in vitro conditions. NMDA administration caused the increase of tracheal smooth muscle response in ovalbumin-induced hyperreactivity to acetylcholine. MK 801 as well as AP-5 provoked the decrease of reactivity mainly to acetylcholine in tracheal smooth muscle, while the former, non-competitive antagonist was more effective. We recorded more pronounced response in tracheal than in lung tissue smooth muscle with more considerable response to acetylcholine than to histamine. The results of experiments show the modification of airway smooth muscles responses by agents modulating the activity of NMDA receptors. They confirm the possibility of NMDA receptors participation in experimental airway hyperreactivity. The results enlarge information regarding the link of the inflammatory diseases and glutamatergic system.     

An operational concept for long-term cinemicrography of cells in mono- and co-culture under highly controlled conditions--the SlideObserver

Cell morphology, proliferation and motility, as well as mono- and heterotypic cell-to-cell interactions, are of increasing interest for in vitro experiments. However, tightly controlling culture conditions whilst simultaneously monitoring the same set of cells is complicated. Moreover, video-microscopy of distinct cells or areas of cells over a prolonged period of time represents a technical challenge. The SlideObserver was designed for cinemicrography of cells in co-and monoculture. The core elements of the system are the SlideReactors, miniaturised hollow fibre-based bioreactors operated in closed perfusion loops. Within the SlideReactors, cells can be cultured under adaptable conditions as well as in direct- and indirect co-culture. The independent perfusion loops enable controlled variation of parameters such as medium, pH, and oxygenation. A combined automated microscope stage and camera set-up allows for micrograph acquisition of multiple user-defined regions of interest within the bioreactor units. For proof of concept, primary cells (HUVEC, human hepatocytes) and cell lines (HuH7, THP-1) were cultured under stable and varying culture conditions, as well as in mono- and co-culture. The operational system enabled non-stop imaging and automated control of process parameters as well as elective manipulation of either reactor. As opposed to non-perfused culture systems or comparable devices for cinemicrographic analysis, the SlideObserver allows simultaneous morphological monitoring of an entire culture of cells in multiple bioreactors.     

Surprising spectra of root-associated fungi in submerged aquatic plants

Similarly to plants from terrestrial ecosystems, aquatic species harbour wide spectra of root-associated fungi (RAF). However, comparably less is known about fungal diversity in submerged roots. We assessed the incidence and diversity of RAF in submerged aquatic plants using microscopy, culture-dependent and culture-independent techniques. We studied RAF of five submerged isoetid species collected in four oligotrophic freshwater lakes in Norway. Levels of dark septate endophytes (DSE) colonization differed among the lakes and were positively related to the organic matter content and negatively related to pH. In total, we identified 41 fungal OTUs using culture-dependent and culture-independent techniques, belonging to Mucoromycotina, Chytridiomycota, Glomeromycota, Ascomycota as well as Basidiomycota. Sequences corresponding to aquatic hyphomycetes (e.g. Nectria lugdunensis, Tetracladium furcatum and Varicosporium elodeae) were obtained. Eight arbuscular mycorrhizal taxa belonging to the orders Archaeosporales, Diversisporales and Glomerales were also detected. However, the vast majority of the fungal species detected (e.g. Ceratobasidium sp., Cryptosporiopsis rhizophila, Leptodontidium orchidicola, and Tuber sp.) have previously been known only from roots of terrestrial plants. The abundance and phylogenetic distribution of mycorrhizal as well as nonmycorrhizal fungi in the roots of submerged plants have reshaped our views on the fungal diversity in aquatic environment.     

Assessment of the working range and effect of sodium dichloroisocyanurate on Pseudomonas aeruginosa biofilms and planktonic cells

Sodium dichloroisocyanurate (NaDCC) is a chemical agent that acts against microorganisms in a manner similar to that of sodium hypochlorite by releasing free available chlorine. NaDCC has been approved by the WHO for the emergency treatment of water and by the US EPA for routine treatment of water. Previous studies assessing the effectiveness of NaDCC for the treatment of water implied that NaDCC should have a wide array of disinfecting effects beyond the treatment of planktonic cells in potable water. In this study the biocidal effects of NaDCC against Pseudomonas aeruginosa cells in different growth modes including planktonic cells and biofilms were explored. The data showed that a 60% dilution of the standard NaDCC solution was effective in the treatment of both P. aeruginosa planktonic cells and biofilms.     

Rac signaling in breast cancer: a tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.     

The non-proteinogenic amino acids l-methionine sulfoximine and dl-phosphinothricin activate mTOR

l-Methionine sulfoximine (MSO) and dl-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.     

Higher nitrate-reducer diversity in macrophyte-colonized compared to unvegetated freshwater sediment

Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification–denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.     

Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.