Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.     

Limited conformational change of beta-lactoglobulin when adsorbed at the air-water interface

Detailed insight can be obtained from proteins at and near the air-water interface using external reflection IR and circular dichroism techniques. Besides information on local protein concentrations and surface layer thickness, it is shown that beta-lactoglobulin displays a limited unfolding at the interface. The conformational change is comparable to that observed upon heat-induced aggregation of the protein and can be understood in view of the high surface concentration of the protein (approximately 40% volume fraction). The layer thickness and the conformational properties of the protein do not depend on the bulk concentration. After adsorption of beta-lactoglobulin to a preformed lipid monomolecular layer a similar conformational change is induced, suggesting that the folding properties of the protein itself determine the extent of conformational changes at the interfaces.     

Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).     

Boosting for tumor classification with gene expression data

MOTIVATION: Microarray experiments generate large datasets with expression values for thousands of genes but not more than a few dozens of samples. Accurate supervised classification of tissue samples in such high-dimensional problems is difficult but often crucial for successful diagnosis and treatment. A promising way to meet this challenge is by using boosting in conjunction with decision trees. RESULTS: We demonstrate that the generic boosting algorithm needs some modification to become an accurate classifier in the context of gene expression data. In particular, we present a feature preselection method, a more robust boosting procedure and a new approach for multi-categorical problems. This allows for slight to drastic increase in performance and yields competitive results on several publicly available datasets. AVAILABILITY: Software for the modified boosting algorithms as well as for decision trees is available for free in R at http://stat.ethz.ch/~dettling/boosting.html.     

Triggering cell mitosis in higher eukaryotes

Dramatic changes of cell organisation occur at onset of mitosis. Genetic analysis of fission yeast and physiological studies of vertebrate and invertebrate oocytes showed that activation of cyclin B-cdc2 kinase triggers mitosis. Nevertheless, upstream mechanisms responsible for this activation remain largely unknown in somatic cells of higher eukaryotes. This review discusses possible pathways and mechanisms involved in triggering onset of mitosis in such cells, including inhibitory checkpoint mechanisms that detect defects in structural organisation of the cell.