Expression of TGF-beta type II receptor antisense RNA impairs TGF-beta signaling in vitro and promotes mammary gland differentiation in vivo

NRC publication: Ye

Quantification of diarrhetic shellfish toxins and identification of novel protein phosphatase inhibitors in marine phytoplankton and mussels

Liquid chromatography (LC)-linked protein phosphatase 1/2A (PP-1/PP2A) bioassay was used to quantitatively identify diarrhetic shellfish toxins in marine phytoplankton (cultured and natural assemblages) and commercially available mussels. Using this approach, multiple protein phosphatase inhibitor profiles of varying composition were found in diarrhetic mussels from Holland and Canada. Based on LC elution positions and relative activity versus PP-1 and PP-2A, at least six inhibitors distinct from known diarrhetic shellfish toxins were identified and termed mussel phosphatase inhibitor (MPI) 19,22,23,25,33 and 42. The levels of these inhibitors, in okadaic acid equivalent units, varied from 100 pg to 3350 ng per g shellfish tissue. The combined levels of PP-1/2A inhibitors in all instances superseded that of okadaic acid/dinophysistoxin-1 and may contribute to the diarrhetic shellfish toxin profile of the contaminated mussels. The efficacy of LC-protein phosphatase bioassay was established for cultured phytoplankton where picogram levels of okadaic acid could be detected from microgram extracts of Prorocentrum lima. Analyses of plankton net tows from estuarine mussel culture sites in Eastern Canada revealed a heterogeneous population of protein phosphatase inhibitors, with dinophysistoxin-1 being most prevalent. This toxin was predominant for at least 2 months in mussel populations in the immediate vicinity of plankton sampling sites. The results are consistent with a hypothetical model in which marine bacteria, cyanobacteria and dinoflagellates combine to produce a variety of protein phosphatase inhibitors effective against signal transduction pathways in higher eukaryotes.Peer reviewed: YesNRC publication: Ye

Growth response of the three cell lines to recombinant human epidermal growth factor (hEGF), heregulin (HRG)-β, and transforming growth factor (TGF)-β

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> [H]thymidine incorporation measured in BRI-JM01, BRI-JM04, and BRI-JM05 cell lines after 24 hours of exposure to serial dilutions of hEGF, HRG-β, or TGF-β. Experiments were conducted in quadruplicate and repeated three times with similar results (for details see text). Western blot analysis of whole cell lysates (20 μg total protein/lane) evaluating ErbB-1, ErbB-2, ErbB-3, and ErbB-4 expression. Additional ErbB-2/Neu immunoprecipitations showed this receptor to be highly phosphorylated in the BRI-JM04 and BRI-JM05 cells. Affinity cross-link labeling of the TGF-β type I, II, III receptors expressed on the BRI-JM01, BRI-JM04 and BRI-JM05 cells using [I]TGF-βin the absence (-) or presence (+) of 100× excess of unlabeled TGF-β. Enzymatic deglycosylation using both heparinitase I/II/III and chondroitinase ABC collapses the diffusely migrating TGF-β type III receptor band to a core protein (approximately 115 kDa, indicated as TβRIII**) labeled with either [I]TGF-βor [I]TGF-β. TGF-β responsive Mv1Lu cells served as a positive control in the TGF-β growth assay and the cell surface cross-link affinity labeling studies. RT-PCR analysis of BRI-JM01 mRNA using two different primer sets (set 1, set 2) confirmed the presence of TGF-β type III receptor transcripts in the BRI-JM01 cells. TGF-β type III receptor-expressing BRI-JM05 cells (part e) were used as a positive control for TGF-β type III receptor expression, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Experiments were repeated twice with similar results

Transforming growth factor (TGF)-βinduces an epithelial-to-mesenchymal transition (EMT) in the BRI-JM01 cell line

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Immunofluorescence microscopy demonstrating the altered localization of E-cadherin, N-cadherin and β-catenin, the rearrangement of F-actin and vimentin filaments, and the disappearance of ZO-1 that accompany the TGF-β1 induced EMT (see text for details). Flow cytometry of nonfixed live cells monitoring the presence of E-cadherin at the cell surface of BRI-JM01 cells that were exposed to TGF-βfor up to 120 hours. Nonspecific isogenic IgG (HA) and cells grown in the absence of TGF-β(CTL) were used as a negative and positive control, respectively. Western blot analysis determining the total cellular content of E-cadherin, N-cadherin and β-catenin in BRI-JM01 cells cultured for 24, 72 and 120 hours in the absence (CTL) or presence of TGF-β(see text for details). BRI-JM01 cells were treated with TGF-βfor 48 hours (BRI-JM01T) and passaged several times in the absence of this growth factor. When seeded in Matrigel these 'TGF-βprimed' BRI-JM01 cells formed larger three-dimensional structures than did the nontreated cells

Evaluation of the activated Neu and transforming growth factor-β type II receptor antisense RNA (TβRII-AS) gene expression in mouse mammary tumor virus (MMTV) transgenic mouse mammary tumors and the derived cell lines

<p><b>Copyright information:</b></p><p>Taken from "Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition"</p><p>Breast Cancer Research 2004;6(5):R514-R530.</p><p>Published online 6 Jul 2004</p><p>PMCID:PMC549171.</p><p>Copyright © 2004 Lenferink et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Activated Neu and TβRII-AS transgene expression was determined by RT-PCR in four independent MMTV/activated Neu + TβRII-AS mammary tumors (left panel), in one MMTV/activated Neu mammary tumor (right panel), and the BRI-JM01, BRI-JM04 and BRI-JM05 cell lines. First strand reactions were carried out in the presence or absence of reverse transcriptase (± RT). Genomic DNA obtained from the BRI-JM01, BRI-JM04, and BRI-JM05 cell lines was evaluated by PCR for the presence of the TβRII-AS (middle) and activated Neu (right) transgenes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as positive control (parts a and c, left panels)

A direct interaction between transforming growth factor (TGF)-betas and amyloid-beta protein affects fibrillogenesis in a TGF-beta receptor-independent manner.

Transforming growth factor-beta (TGF-beta) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-beta protein (Abeta) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-beta receptors. Surprisingly, TGF-beta potentiated the neurotoxic effects of the 40-residue Abeta peptide, Abeta-(1-40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-beta and Abeta-(1-40). Surface plasmon resonance confirmed that TGF-beta binds with high affinity directly to Abeta-(1-40) and electron microscopy revealed that TGF-beta enhances Abeta-(1-40) oligomerization. Immunohistochemical examination of mouse brain revealed that hippocampal CA1 and dentate gyrus, two regions classically associated with Abeta-mediated pathology, lack TGF-beta Type I receptor immunoreactivity, thus indicating that TGF-beta receptor-mediated signaling would not be favored in these regions. Our observations not only provide for a unique, receptor-independent mechanism of action for TGF-beta, but also help to reconcile the literature interpreting the role of TGF-beta in Abeta function. These data support a critical etiological role for this mechanism in neuropathological amyloidoses

Assisted Design of Antibody and Protein Therapeutics (ADAPT)

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above