Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

Inter-Relationship between Rhinitis and Conjunctivitis in Allergic Rhinoconjunctivitis and Associated Risk Factors in Rural UK Children

Objective

Allergic conjunctivitis (AC) is a common condition, especially in childhood. The extent to which it occurs concurrently with or independently from allergic rhinitis (AR) has not been well described.

Aim

To examine the inter-relationship between rhinitis and conjunctivitis and the epidemiological risk factors for these conditions in a rural UK population.

Methods

Cross-sectional study of rural school children (aged 5–11 years). Parental questionnaires were used to diagnose allergic outcomes (including conjunctivitis, rhinitis and rhinoconjunctivitis), and to collect data on atopic history, demographic and environmental exposures. Odds ratios of allergic outcome by exposure were examined adjusted for age, sex, breastfeeding, family history of allergy, number of older and younger siblings.

Results

Prevalence of conjunctivitis was 17.5%, rhinitis 15.1% and rhinoconjunctivitis 13.0%. Seasonality of symptoms varied by condition: 64.7% of those with conjunctivitis had seasonal symptoms (April-Sept only), 46.7% of those with rhinitis and 92.2% of those with rhinoconjunctivitis. Living on a farm consistently reduced the risk of conjunctivitis (odds ratio 0.47, 95%CI 0.29–0.79, p = 0.004), rhinitis (OR 0.57, 95%CI 0.33–1.01, p = 0.05) and rhinoconjunctivitis (OR 0.57, 95%CI 0.32–1.03, p = 0.06). Exposure to farm animals (particularly in early life), current consumption of unpasteurised milk and playing in a barn or stable significantly reduced the risk of all three conditions.

Conclusion

More children had parent-reported conjunctivitis than rhinitis. The majority of children with either condition also reported symptoms with the other condition. Farmers’ children have less eye and/or nasal symptoms. A number of farming variables linked with the farm microbial environment are likely to be mediating the protective effect.     

Two fluorogenic substrates for purine nucleoside phosphorylase,selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

Population genetic structure of the European kestrel Falco tinnunculus in Central Poland

We analysed the genetic structure of the European kestrel population of Central Poland using nine highly polymorphic microsatellite loci. Samples were collected in two urban locations (Warsaw and ?ód?) and two rural areas. Sampling locations were at nearly equal distances from each other along an east to west line. We performed genotyping in a total of 99 birds. The results revealed the presence of a genetic structure in the population investigated. Bayesian clustering indicated that samples originated from more than one population. Genetic differentiation was less pronounced among the birds nesting in Warsaw and in the two rural sites, whereas all pairwise comparisons with the ?ód? population indicated moderate and significant genetic differentiation. The observed pattern of differentiation might have been caused by two factors: changes in allele frequency between seasons and/or the founding of the urban population of ?ód? from a different source population than the urban population from Warsaw. Additionally, we found a rather high gene flow among kestrels from the Warsaw urban area and the two investigated rural areas.     

Intestinal microflora of autistic children

Autistic behavior is often accompanied by numerous disturbing symptoms on the part of gastrointestinal system, such as abdominal pain, constipation or diarrhea. These problems are often connected with deregulation of physiological microflora in intestine. The aim of this study was to determine differences in intestinal microflora of autistic and healthy children. Strains of Clostridium spp. and enterococci were isolated more frequently from stool samples of autistic children and rarely lactobacilli. Quantitative differences were observed maliny among staphylococci, Candida spp. and Clostridium perfringens. Monitoring and stabilization of intestinal microflora and knowledge about role of particular strains in etiology of autistic disorders can increase the chances for appropriate therapy.     

Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice

Background

Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.

Methods

Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.

Results

Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.

Conclusion

Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.     

Pigment epithelium-derived factor in ulcerative colitis: possible relationship with disease activity

OBJECTIVES: Pigment epithelium-derived factor (PEDF) is an endogenous most potential angiogenic inhibitor and increased expression of PEDF in intestinal mucosa specimens was shown in the course of ulcerative colitis (UC). The aim of the present study was to evaluate serum concentration of pigment epithelium-derived growth factor, a potent anti-angiogenic factor and its possible association with vascular endothelial growth factor (VEGF) levels and disease activity. METHODS: Concentrations of PEDF and VEGF were measured in sera of 33 patients (13 females and 20 males) with active UC. RESULTS: There was significant increase of serum PEDF (32.3+/-2.9 vs. 20.6+/-4.7 ng/mL, P<0.05) as well as VEGF (326.4+/-58.1 vs. 110.9+/-15.7 pg/mL, P<0.05) in UC patients compared to healthy controls. Serum PEDF showed strong, positive correlation with endoscopic score (r=0.622, P<0.001), while such association was absent in respect to VEGF (r=0.05, P=0.77). In contrast serum VEGF decreased in severe UC comparing to patients with a mild course of disease, however the difference was not significant (274.9+/-64.9 vs. 360.4+/-103.4 pg/mL, P=0.53). CONCLUSIONS: Increase in serum PEDF during UC, especially in severe forms of disease suggests its involvement in UC pathogenesis.     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.