Effect of N-cadherin misexpression by the mammary epithelium in mice

N-cadherin is not typically expressed by epithelial cells. However, it is detected in breast cancers and increases tumor cell migration and invasion in vitro. To explore its misexpression, we generated transgenic mice with N-cadherin in the mammary epithelium. Mammary glands appeared normal and no tumors arose spontaneously. To investigate N-cadherin misexpression in mammary tumors, neu was overexpressed through breeding. Tumors developed in +/neu and N-cadherin/neu mice, although few tumors in bitransgenic mice expressed N-cadherin, and they did not differ from N-cadherin-negative tumors.     

Effect of culture conditions on microorganism identification by matrix-assisted laser desorption ionization mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem. 72:1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.     

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA(1-128)), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA(1-76)), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA(7-40) and bCgA(47-70) with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA(7-57), bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60-150 mm Hg, the rat peptide (rCgA(7-57)) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin). Neither Rh-bCgA(7-40) nor Rh-bCgA(47-70) stained the endothelial layer, while Rh-bCgA(47-70) but not Rh-bCgA(7-40) stained the smooth muscle compartment. Analogously, bCgA(47-66) but not bCgA(7-40) reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA(7-57) and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA(47-66)), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.     

Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.     

Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, beta-tubulin, and LSU rDNA

To provide a robust phylogeny of Pezizaceae, partial sequences from two nuclear protein-coding genes, RPB2 (encoding the second largest subunit of RNA polymerase II) and beta-tubulin, were obtained from 69 and 72 specimens, respectively, to analyze with nuclear ribosomal large subunit RNA gene sequences (LSU). The three-gene data set includes 32 species of Peziza, and 27 species from nine additional epigeous and six hypogeous (truffle) pezizaceous genera. Analyses of the combined LSU, RPB2, and beta-tubulin data set using parsimony, maximum likelihood, and Bayesian approaches identify 14 fine-scale lineages within Pezizaceae. Species of Peziza occur in eight of the lineages, spread among other genera of the family, confirming the non-monophyly of the genus. Although parsimony analyses of the three-gene data set produced a nearly completely resolved strict consensus tree, with increased confidence, relationships between the lineages are still resolved with mostly weak bootstrap support. Bayesian analyses of the three-gene data, however, show support for several more inclusive clades, mostly congruent with Bayesian analyses of RPB2. No strongly supported incongruence was found among phylogenies derived from the separate LSU, RPB2, and beta-tubulin data sets. The RPB2 region appeared to be the most informative single gene region based on resolution and clade support, and accounts for the greatest number of potentially parsimony informative characters within the combined data set, followed by the LSU and the beta-tubulin region. The results indicate that third codon positions in beta-tubulin are saturated, especially for sites that provide information about the deeper relationships. Nevertheless, almost all phylogenetic signal in beta-tubulin is due to third positions changes, with almost no signal in first and second codons, and contribute phylogenetic information at the "fine-scale" level within the Pezizaceae. The Pezizaceae is supported as monophyletic in analyses of the three-gene data set, but its sister-group relationships is not resolved with support. The results advocate the use of RPB2 as a marker for ascomycete phylogenetics at the inter-generic level, whereas the beta-tubulin gene appears less useful.     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

Cytokines and lymphocyte proliferation in patients with different clinical forms of chromoblastomycosis

Chromoblastomycosis is a chronic, often debilitating, suppurative, granulomatus mycosis of the skin and subcutaneous tissues beginning after inoculation trauma. It occurs world-wide, but is more frequently observed in tropical countries such as Brazil. The disease is usually insidious, and the lesions increase slowly but progressively, not responding to the usual treatments and quite often reappearing. The host defense mechanism in chromoblastomycosis has not been extensively investigated. Some studies have focused on fungus-host interaction, showing a predominantly cellular immune response, with the activation of macrophages involved in fungus phagocytosis. Although phagocytosis did occur, death of fungal cells was rarely observed. The ability of Fonsecaea pedrosoi to produce secreted or cell wall-associated melanin-like components, protects against destruction by host immune cells in vitro. Until now, the T cell immune response in chromoblastomycosis is undefined. In the present work, it was shown that, in patients with the severe form of the disease, predominant production of IL-10 cytokine, low levels of IFN-gamma and inefficient T cell proliferation were induced. In contrast, in patients with a mild form of the disease, predominant production of IFN-gamma cytokine, low levels of IL-10 and efficient T cell proliferation were observed.     

Survival and persistence of opportunistic Burkholderia species in host cells

Burkholderia are microorganisms that have a unique ability to adapt and survive in many different environments. They can also serve as biopesticides and be used for the biodegradation of organic compounds. Usually harmless while living in the soil, these bacteria are opportunistic pathogens of plants and immunocompromised patients, and occasionally infect healthy individuals. Some of the species in this genus can also be utilised as biological weapons. They all possess very large genomes and have two or more circular chromosomes. Their survival and persistence, not only in the environment but also in host cells, offers a remarkable example of bacterial adaptation.