Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Metal free carboamination of internal alkynes--an easy access to polysubstituted quinolines.

A metal free carboamination of unactivated alkynes towards highly substituted quinolines was realized in the presence of a synergistic Brønsted acid catalyst system. Supported by mechanistic probes, the reaction proceeds via a highly reactive vinyl cation in a C-C bond formation--Schmidt reaction sequence. The irreversible extrusion of N2, as a powerful driving force, allows for a general conversion of poorly nucleophilic aliphatic alkynes.

Kelvin probe force microscopy study on conjugated polymer/fullerene bulk heterojunction organic solar cells.

We conducted a comprehensive Kelvin probe force microscopy (KPFM) study on a classical organic solar cell system consisting of MDMO-PPV/PCBM blends. The KPFM method yields the information of topography and local work function at the nanometer scale. Experiments were performed either in the dark or under cw laser illumination at 442 nm. We identified distinct differences in the energetics on the surface of chlorobenzene and toluene cast blend films. Together with high-resolution scanning electron microscopy (SEM) experiments we were able to interpret the KPFM results and to draw some conclusions for the electron transport toward the cathode in the solar cell configuration. The results suggest that surfaces of toluene cast films exhibit a morphologically controlled hindrance for electron propagation toward the cathode, which is usually evaporated on top of the films in the solar cell device configuration.

A new calcium-sensor based on ion-selective conductometric microsensors--membranes and features.

Based on the concept of ion-selective conductometric microsensors (ISCOM) a new calcium sensor was developed and characterized. ISCOM have a single probe, all-solid-state construction and do not need a reference electrode. These sensors are amenable to miniaturization and integration in the true sense of integrated circuit and microsystem technologies. The detection is accomplished by measurement of the bulk conductance Gm of a thin polymeric membrane containing an ion-complexing agent, where the magnitude of Gm can be related to the content of the primary ion in the analyzed solution. Thin-film platinum electrodes forming an interdigitated electrode are used as the transducer to detect the conductivity of the polymeric membrane. Optimization of the membrane composition was carried out by testing different types of calcium-ionophores, polymers, and plasticizers. The sensor characteristics have been investigated. The limit of detection is about 10(-7) mol L(-1). The dynamic range is 10(-6)-10(-1) mol L(-1) with a response time of less than 5 s. These parameters are comparable to those of corresponding potentiometric calcium selective electrodes (ISE). The Ca(2+)-ISCOM demonstrates good practical relevant selectivities against typical interfering ions for biomedical and environmental applications.

Exploring local and non-local interactions for protein stability by structural motif engineering.

In order to probe the relative contribution of local and non-local interactions to the thermodynamic stability of proteins, we have devised an experimental approach based on a combination of motif engineering and sequence shuffling. Candidate chain segments in an immunoglobulin V(L) domain were identified whose conformation is proposed to be dominated by non-local interactions. Locally interacting structural motifs of a different conformation were then constructed as replacements, by introducing motif consensus sequences. We find that all nine replacements we constructed systematically reduce the folding cooperativity. By comparing this destabilising effect with the folding transitions of shuffled sequences for three of these motifs, we estimate the contribution of local, native interactions to the free energy of folding. Our results suggest that local and non-local interactions contribute to stability by an approximately equal amount, but that local interactions stabilise by increasing the resistance to denaturation while non-local interactions increase folding cooperativity. The systematic loss of stability by sequence shuffling in these host-guest experiments suggests that the designed interactions indeed are present in the native state, thus consensus sequence engineering may be a useful tool in structure design, but non-local interactions must be taken into account for global stability engineering. Statistical approaches are powerful tools for engineering protein structure and stability, but an analysis based on local sequence propensities alone does not adequately represent the balance of sequence and context in protein structures.

Absence of an unusual "densely methylated island" at the hamster dhfr ori-beta.

An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-beta locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G1, S, or G2 + M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines (mC) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of mC residues that complements the more commonly used hydrazine method. These methods were capable of detecting mC in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-beta DMI was not detected in any of these cell populations using any of these methods.