Annelated pyrrolo-pyrimidines from amino-cyanopyrroles and BMMAs as leads for new DNA-interactive ring systems

The efficient one-pot synthesis of several new tricyclic systems of type 1 and 2, obtained from the reaction of substituted 2-amino-3-cyanopyrroles and 3-amino-4-cyanopyrroles with BMMAs, is reported. The duration and yields of the reaction strongly depend on the reactivity of the starting pyrrole and on the size of the ring to be formed. Mechanist features of the reaction were investigated and proposed by studying also the reactivity of a 3-aminopyrrole-2,4-dicyano substituted. The method reported represents the first example of the use of BMMA reagents in combination with pyrrole derivatives and allows an easy and versatile entry to a large number of hitherto unknown pyrrolo-pyrimidines further annelated with nitrogen heterocycles of different sizes. These new polycondensed heterocycles possess the requisite to interact with DNA.     

Finishers and nonfinishers in the 'Swiss Cycling Marathon ' to qualify for the 'Race Across America '

Knechtle, B, Knechtle, P, Rüst, CA, Rosemann, T, and Lepers, R. Finishers and nonfinishers in the 'Swiss Cycling Marathon' to qualify for the 'Race across America.' J Strength Cond Res 25(12): 3257-3263, 2011-We compared the characteristics of prerace anthropometry, previous experience, and training and support during the race in 39 finishers and 37 nonfinishers in the 'Swiss Cycling Marathon,' over 720 km. In this race, the cyclists intended to qualify for the 'Race across America,' the longest nonstop cycling race in the World from the West to the East of the USA. Finishers in the 'Swiss Cycling Marathon' had a lower body mass, a lower body mass index, lower circumferences of upper arm and thigh, a lower percent body fat, completed more weekly training units, covered more kilometers in the longest training ride, rode at a faster speed during training, rode more kilometers per week and for more hours, had more previous finishes in the 'Swiss Cycling Marathon' and a lighter race bike compared to the nonfinishers. In the bivariate analysis, the cycling distance per training unit (r = 0.37), the duration per training unit (r = 0.44), the speed per training unit (r = -0.59), using nutrition provided by the organizer (r = 0.50), and using own nutrition (r = 0.49) during the race were significantly and positively associated with race time. For practical applications, anthropometric characteristics such as a low body mass or low body fat were not related to race time, whereas training characteristics and nutrition during the race were associated with race time. The key to a successful finish in an ultraendurance cycling race such as the 'Swiss Cycling Marathon' seems a high speed in training and an appropriate nutrition during the race.     

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.     

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.     

A retinal proteomics-based study identifies αA-crystallin as a sex steroid-regulated protein

Sex steroids influence the structural and functional organization of ocular tissues, promote survival in several pathological conditions including retinal neurodegeneration and have a prominent role in age-related eye diseases as well as neurodegenerative diseases. However, their underlying mechanisms are still elusive. We explored proteomic profiling of rat retinas following intravitreal injection of the bioactive 17β-estradiol or androgen dihydrotestosterone. Using narrow range 2-DE gels and MALDI-TOF-MS analysis, we identified three sex steroid-regulated proteins: the galectin-related-inter-fiber (GRIFIN) which is a galectin family member protein of unknown function, the fatty acid-binding protein epidermal-5 (FABP5) protein responsible for the fatty acid uptake and transport and the small heat shock αA-crystallin (CRYAA) protein involved in preventing aggregation of denatured or unfolded proteins. Changes in the expression of these proteins revealed a predominant estrogenic effect and the multiple CRYAA protein species reflected posttranslational modifications. Sex steroid-mediated modifications of CRYAA were confirmed by Western blotting analysis. This study provides new target proteins for sex steroids with a potential link to age-related diseases associated with proteotoxic stress.     

Murine melanoma-infiltrating dendritic cells are defective in antigen presenting function regardless of the presence of CD4CD25 regulatory T cells

Tumor-infiltrating dendritic cells are often ineffective at presenting tumor-derived antigen in vivo, a defect usually ascribed to the suppressive tumor environment. We investigated the effects of depleting CD4⁺CD25⁺ “natural” regulatory T cells (Treg) on the frequency, phenotype and function of total dendritic cell populations in B16.OVA tumors and in tumor-draining lymph nodes. Intraperitoneal injection of the anti-CD25 monoclonal antibody PC61 reduced Treg frequency in blood and tumors, but did not affect the frequency of tumor-infiltrating dendritic cells, or their expression of CD40, CD86 and MHCII. Tumor-infiltrating dendritic cells from PC61-treated or untreated mice induced the proliferation of allogeneic T cells in vitro, but could not induce proliferation of OVA-specific OTI and OTII T cells unless specific peptide antigen was added in culture. Some proliferation of naïve, OVA-specific OTI T cells, but not OTII T cells, was observed in the tumor-draining LN of mice carrying B16.OVA tumors, however, this was not improved by PC61 treatment. Experiments using RAG1^−/− hosts adoptively transferred with OTI and CD25-depleted OTII cells also failed to show improved OTI and OTII T cell proliferation in vivo compared to C57BL/6 hosts. We conclude that the defective presentation of B16.OVA tumor antigen by tumor-infiltrating dendritic cells and in the tumor-draining lymph node is not due to the presence of “natural” CD4⁺CD25⁺ Treg.     

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus

Background

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

Methodology/Principal Findings

We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8⁺ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP_CTL). MCMV/ZEBOV-NP_CTL induced high levels of long-lasting (>8 months) CD8⁺ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.

Conclusions/Significance

This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.     

The influence of calcium ions on nickel modulation of NMDA receptor currents

N-Methyl-D-aspartate (NMDA) receptors (NRs) are glutamate-gated channels critical for the functioning of the nervous system. They are assembled from two types of subunits, the essential GluN1 and at least one type of GluN2 (A, B, C, D) subunit. Nickel (Ni) modulates the NR current in a way dependent on the GluN2 subunit present. Besides voltage-dependent and voltage-independent inhibition, in GluN2B-containing channels Ni enhances channel activity. We have recently identified several domains of the channel involved in Ni interaction, but many aspects of this modulation remain elusive. The purpose of the present work is to investigate the role of calcium (Ca) in the effect of Ni on the NR current measured by voltage- and patch-clamp in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells expressing GluN1/GluN2B recombinant receptors. In both expression systems, in the presence of a physiological concentration of Ca (1.8 mM), Ni increased the NR current with EC(50) in the μM range, but this potentiation was reduced by decreasing Ca concentration or when Ca was substituted with Ba. In injected oocytes, the effect of Ni in 0.3 mM external Ba was only inhibitory (IC(50) = 65 μM). Increasing the internal calcium buffering by EGTA and BAPTA application, as well as incubation with cytoskeleton perturbing agents, colchicine and cytochalasin-D, did not produce major modifications in the Ni effect. These observations indicate that Ni-mediated potentiation is not dependent on Ca influx and internal Ca concentration, but it is dependent on external Ca, which possibly interacts with the extracellular portion of the protein through a modulatory binding site.     

Polarized endocytosis of the keratinocyte growth factor receptor in migrating cells: role of SRC-signaling and cortactin

Cell migration is a physiological process that requires endocytic trafficking and polarization of adhesion molecules and receptor tyrosine kinases (RTKs) to the leading edge. Many growth factors are able to induce motility by binding to specific RTK on target cells. Among them, keratinocyte growth factor (KGF or FGF7) and fibroblast growth factor 10 (FGF10), members of the FGF family, are motogenic for keratinocytes, and exert their action by binding to the keratinocyte growth factor receptor (KGFR), a splicing variant of FGFR2, exclusively expressed on epithelial cells. Here we analyzed the possible role of cortactin, an F-actin binding protein which is tyrosine phosphorylated by Src and is involved in KGFR-mediated cell migration, in the KGFR endocytosis and polarization to the leading edge of migrating cells upon ligand-induced stimulation. Biochemical phosphorylation study revealed that both KGF and FGF10 were able to induce tyrosine phosphorylation of Src and in turn of cortactin, as demonstrated by using the specific pharmacological Src-inhibitor SU6656, although FGF10 effect was delayed with respect to that promoted by KGF. Immunofluorescence analysis demonstrated the polarized localization of KGFR upon ligand stimulation to the leading edge of migrating keratinocytes, process that was regulated by Src. Moreover, we showed that the colocalization of cortactin with KGFR at the plasma membrane protrusions and on early endosomes after KGF and FGF10 treatment was Src-dependent. Further, by using a RNA interference approach through microinjection, we showed that cortactin is required for KGFR endocytosis and that the clathrin-dependent internalization of the receptor is a critical event for its polarization. Finally, KGFR expression and polarization enhanced cell migration in a scratch assay. Our results indicate that both Src and cortactin play a key role in the KGFR endocytosis and polarization at the leading edge of migrating keratinocytes, supporting the crucial involvement of RTK trafficking in cell motility.