Characterization of dual agonists for kinin B1 and B2 receptors and their biased activation of B2 receptors

Kinin B1 and B2 receptors (kB1R and kB2R) play important roles in many physiological and pathological processes. In some cases, kB1R or kB2R activation can have overlapping or complementary beneficial effects, thus an activator of both receptors might be advantageous. We found that replacement of the C-terminal Arg in the natural kB2R activators bradykinin (BK) or kallidin (KD) with Lys (K(9)-BK or K(10)-KD) resulted in agonists that effectively stimulate the downstream signaling of both the kB1R and kB2R as measured by increased inositol turnover, intracellular calcium, ERK1/2 phosphorylation, arachidonic acid release and NO production. However, K(9)-BK and K(10)-KD displayed some characteristics of biased agonism for kB2Rs as indicated by the rapid kinetics of ERK1/2 phosphorylation induced by K(9)-BK or K(10)-KD compared with the prolonged response mediated by BK or KD. In contrast, kinetics of ERK phosphorylation stimulated by K(10)-KD activation of the kB1R was the same as that induced by known kB1R agonist des-Arg(10)-KD. Furthermore, the endocytosis of kB2Rs mediated by K(9)-BK and K(10)-KD was remarkably less than that induced by BK and KD respectively. K(10)-KD stimulated kB1R and kB2R-dependent calcium responses and ERK1/2 phosphorylation in bovine endothelial cells. In cytokine-treated human endothelial cells, K(10)-KD stimulated ERK1/2 phosphorylation and a transient peak of NO production that was primarily kB2R-dependent. K(10)-KD also stimulated prolonged NO production that was both kB1R and kB2R-dependent. These data provide the first examples of dual agonists of kB1R and kB2R, and a biased agonist of kB2R and may provide useful clues for developing dual modulators of kB1Rs and kB2Rs for potential therapeutic use.     

Jan Evangelista Purkyně/Purkinje (1787–1869) and the establishment of cellular physiology—Wrocław/Breslau as a central European cradle for a new science

Being a professor of physiology in Wroc?aw/Breslau till the half of nineteenth century, Jan Evangelista Purkyně/Purkinje made, along with his students, many crucial discoveries combining original experimental approaches with new advanced microscopy and histology techniques. Here, he established first Institute of Physiology worldwide and created a framework for the new science of cellular physiology. With his work, he not only substantially contributed to the establishment of cellular and protoplasmic concepts in biology but represented a rare type of Central European scholar by bridging communities separated by ethnicity and language     

Safety and efficacy of fluticasone/formoterol combination therapy in adolescent and adult patients with mild-to-moderate asthma: a randomised controlled trial

Background

Early wheezing and asthma are relevant health problems in the tropics. Mite sensitization is an important risk factor, but the roles of others, inherent in poverty, are unknown. We designed a birth-cohort study in Cartagena (Colombia) to investigate genetic and environmental risk factors for asthma and atopy, considering as particular features perennial exposure to mites, parasite infections and poor living conditions.

Methods

Pregnant women representative of the low-income suburbs of the city were randomly screened for eligibility at delivery; 326 mother-infant pairs were included at baseline and biological samples were collected from birth to 24 months for immunological testing, molecular genetics and gene expression analysis. Pre and post-natal information was collected using questionnaires.

Results

94% of families were from the poorest communes of the city, 40% lacked sewage and 11% tap-water. Intestinal parasites were found as early as 3 months; by the second year, 37.9% of children have had parasites and 5.22% detectable eggs of Ascaris lumbricoides in stools (Median 3458 epg, IQR 975-9256). The prevalence of "wheezing ever" was 17.5% at 6 months, 31.1% at 12 months and 38.3% at 24 months; and recurrent wheezing (3 or more episodes) 7.1% at 12 months and 14.2% at 24 months. Maternal rhinitis [aOR 3.03 (95%CI 1.60-5.74), p = 0.001] and male gender [aOR 2.09 (95%CI 1.09 - 4.01), p = 0.026], increased risk for wheezing at 6 months. At 24 months, maternal asthma was the main predisposing factor for wheezing [aOR 3.65 (95%CI 1.23-10.8), p = 0.01]. Clinical symptoms of milk/egg allergy or other food-induced allergies were scarce (1.8%) and no case of atopic eczema was observed.

Conclusions

Wheezing is the most frequent phenotype during the first 24 months of life and is strongly associated with maternal asthma. At 24 months, the natural history of allergic symptoms is different to the "atopic march" described in some industrialized countries. This cohort is representative of socially deprived urban areas of underdeveloped tropical countries. The collection of biological samples, data on exposure and defined phenotypes, will contribute to understand the gene/environment interactions leading to allergy inception and evolution.     

The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box–type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP-triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs, suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP-induced signaling.     

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

ABSTRACT: BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.