Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Focal adhesion kinase is not required for integrin function or viability in Drosophila

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.     

Trypanosoma cruzi heat-shock protein-70 kDa,alone or fused to the parasite KMP11 antigen,induces functional maturation of murine dendritic cells

We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.     

Clinical efficacy of blue light full body irradiation as treatment option for severe atopic dermatitis

Background

Therapy of atopic dermatitis (AD) relies on immunosuppression and/or UV irradiation. Here, we assessed clinical efficacy and histopathological alterations induced by blue light-treatment of AD within an observational, non-interventional study.

Methodology/Principal Findings

36 patients with severe, chronic AD resisting long term disease control with local corticosteroids were included. Treatment consisted of one cycle of 5 consecutive blue light-irradiations (28.9 J/cm²). Patients were instructed to ask for treatment upon disease exacerbation despite interval therapy with topical corticosteroids. The majority of patients noted first improvements after 2–3 cycles. The EASI score was improved by 41% and 54% after 3 and 6 months, respectively (p≤0.005, and p≤0.002). Significant improvement of pruritus, sleep and life quality was noted especially after 6 months. Also, frequency and intensity of disease exacerbations and the usage of topical corticosteroids was reduced. Finally, immunohistochemistry of skin biopsies obtained at baseline and after 5 and 15 days revealed that, unlike UV light, blue light-treatment did not induce Langerhans cell or T cell depletion from skin.

Conclusions/Significance

Blue light-irradiation may represent a suitable treatment option for AD providing long term control of disease. Future studies with larger patient cohorts within a randomized, placebo-controlled clinical trial are required to confirm this observation.     

Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells

Regulatory CD4(+)CD25(+) T cells are important in suppressing immune responses. The requirements for the maintenance of peripheral CD4(+)CD25(+) T cells remain incompletely understood. Receptor activator of NF-kappaB (RANK) and its ligand (RANKL; also known as CD254, OPGL and TRANCE) are key regulators of bone remodeling, mammary gland formation, lymph node development and T-cell/dendritic cell communication. Here we report that RANKL is expressed in keratinocytes of the inflamed skin. RANKL overexpression in keratinocytes resulted in functional alterations of epidermal dendritic cells and systemic increases of regulatory CD4(+)CD25(+) T cells. Thus, epidermal RANKL expression can change dendritic cell functions to maintain the number of peripheral CD4(+)CD25(+) regulatory T cells. Epidermal RANKL mediated ultraviolet-induced immunosuppression and overexpression of epidermal RANKL suppressed allergic contact hypersensitivity responses and the development of systemic autoimmunity. Therefore, environmental stimuli at the skin can rewire the local and systemic immune system by means of RANKL.     

Testing the quick meal hypothesis: The effect of pulp on hoarding and seed predation of Hymenaea courbaril by red‐rumped agoutis (Dasyprocta leporina)

Abstract: Red‐rumped agoutis (Dasyprocta leporina) are important seed dispersers/predators of Neotropical large‐seeded plants. Several species of seeds cached by agoutis have an edible reward, in contrast to temperate rodent‐dispersed diaspores. The quick meal hypothesis states that the presence of a reward such as edible pulp will enhance the efficiency of rodents as seed disperses by satiating the animal and, consequently, reducing seed predation and enhancing hoarding. In this study, this hypothesis was tested using as the reference system the pulp and seeds of Hymenaea courbaril. Seeds with and without pulp were offered to agoutis and the behaviour of each individual was recorded. Since the probability of predation and hoarding were complementary, we used the probability of predation. The proportion of agoutis that preyed on at least one seed was similar for seeds with (42.8% of individuals) and without (40.0% of individuals) pulp. In agoutis that preyed upon at least one seed, the probability that they killed a seed did not differ between seeds with (0.17 ± 0.03) and without (0.20 ± 0.08) pulp. Hence, these results do not support the ‘quick meal hypothesis’.