The chicken neural extracellular matrix molecule restrictin: similarity with EGF-, fibronectin type III-, and fibrinogen-like motifs.

Restrictin is a chick neural extracellular matrix protein implicated in neural cell attachment and found to be associated with the cell surface recognition protein F11. Here we show by cDNA cloning that restrictin is a large multidomain protein composed of 4 structural motifs. At the N-terminus restrictin contains a cysteine-rich segment of about 140 aa that might link restrictin monomers into oligomers. This region is followed by 4.5 epidermal growth factor-like repeats and then by 9 consecutive motifs that are similar to fibronectin type III motifs. At the C-terminus restriction is related to the beta and gamma chains of fibrinogen, including similarity to a calcium-binding segment. Restrictin shows substantial sequence similarity with tenascin (cytotactin) throughout the polypeptide, and like tenascin, it forms oligomeric structures, as revealed by electron microscopy of immunoaffinity-purified restriction. The cell attachment site of restrictin is mapped to the C-terminal region by antibody perturbation experiments.     

Orcein and Litmus

Orcein was separated into 14 dyes by partition chromatography. Their constitutions were determined mainly by spectroscopy and led to formulae that are derived from 7-amino-2-phenoxazone, 7-hydroxy-2-phenoxazone, and 7-amino-2-phenoxazime, and that were confirmed by syntheses. The major constituent of litmus is assembled polymerically from 7-hydroxy-2-phenoxazone chromophores. The mechanism of formation is elucidated.     

Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses

Background  

Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data.     

Effector proteins of the bacterial pathogen Pseudomonas syringae alter the extracellular proteome of the host plant, Arabidopsis thaliana

In plants, potential pathogenic bacteria do not enter the host cell. Therefore, a large portion of the molecular interaction between microbial pathogen and host occurs in the extracellular space. To investigate potential mechanisms of disease resistance and susceptibility, we analyzed changes in the extracellular proteome, or secretome, using the Arabidopsis-Pseudomonas syringae pathosystem. This system provides the possibility to directly compare interactions resulting in basal resistance, susceptibility, and gene-specific resistance by using different genotypes of Pseudomonas on the same host. After infecting suspension-cultured cells of Arabidopsis with the Pseudomonas strain of interest, we isolated protein from the cell culture medium representing the secretome. After one-dimensional gel separation and in-gel digestion of proteins, we used iTRAQ (isobaric tags for relative and absolute quantitation) labeling in conjunction with LC-MS/MS to perform relative quantitative comparisons of the secretomes from each of these interactions. We obtained quantitative information from 45 Arabidopsis proteins that were present in all three biological experiments. We observed complex patterns of accumulation, ranging from proteins that decreased in abundance in the presence of all three bacterial strains to proteins that specifically increased or decreased during only one of the interactions. A particularly intriguing result was that the virulent bacteria (e.g. a susceptible interaction) caused the extracellular accumulation of a specific subset of host proteins lacking traditional signal peptides. These results indicate that the pathogen may manipulate host secretion to promote the successful invasion of plants.     

Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture

In the mammal, the pluripotent cells of embryo differentiate and commit to either the mesoderm/endoderm lineages or the ectoderm lineage during gastrulation. In culture, the ability to direct lineage choice from pluripotent cells into the mesoderm/endoderm or ectoderm lineages will enable the development of technologies for the formation of highly enriched or homogenous populations of cells. Here we show that manipulation of cell:cell contact and a mesoderm suppressing activity in culture affects the outcome of pluripotent cell differentiation and when both variables are manipulated appropriately they can direct differentiation to either the mesoderm or ectoderm lineage. The disruption of cell:cell contacts and removal of a mesoderm suppressor activity results in the differentiation of pluripotent, primitive ectoderm-like cells to the mesoderm lineage, while maintenance of cell:cell contacts and inclusion, within the culture medium, of a mesoderm suppressing activity results in the formation of near homogenous populations of ectoderm. Understanding the contribution of these variables in lineage choice provides a framework for the development of directed differentiation protocols that result in the formation of specific cell populations from pluripotent cells in culture.