Evidence for an intramembrane component associated with a cellulose microfibril-synthesizing complex in higher plants

Freeze-fracture of rapidly frozen, untreated plant cells reveals terminal complexes on E-fracture faces and intramembrane particle rosettes on P-fracture faces. Terminal complexes and rosettes are associated with the ends of individual microfibril impressions on the plasma membrane. In addition, terminal complexes and rosettes are associated with the impressions of new orientations of microfibrils. These structures are sparse within pit fields where few microfibril impressions are observed, but are abundant over adjacent impressions of microfibrils. It is proposed that intramembrane rosettes function in association with terminal complexes to synthesize microfibrils. The presence of a cellulosic microfibril system in Zea mays root segments is confirmed by degradation experiments with Trichoderma cellulase

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component

Modulation of the rat angiotensin type 1a receptor by an upstream short open reading frame

The rat angiotensin type 1a receptor (ATR) is a peptide hormone G protein-coupled receptor (GPCR) that plays a key role in electrolyte homeostasis and blood pressure control. There is a highly conserved short open reading frame (sORF) in exon 2 (E2) that is downstream from exon 1 (E1) and upstream of the ATR coding region located in exon 3 (E3). To determine the role of this E2 sORF in ATR signaling, human embryonic kidney-293 (HEK293) cells were transfected with plasmids containing ATR cDNA with either an intact or disrupted E2 sORF. The intact sORF attenuated the efficacy of angiotensin (Ang) II (p \u3c 0.001) and sarcosine,Ile,Ile-Ang II (SII), (p \u3c 0.01) to activate ATR signaling through extracellular signal-related kinases 1/2 (ERK1/2). A time-course showed agonist-induced ATR-mediated ERK1/2 activation was slower in the presence of the intact compared to the disrupted sORF [Ang II: p \u3c 0.01 and SII: p \u3c 0.05]. Ang II-induced ERK1/2 activation was completely inhibited by the protein kinase C (PKC) inhibitor Ro 31-8220 regardless of whether the sORF was intact or disrupted. Flow cytometric analyses suggested the intact sORF improved cell survival; the percentage of live cells increased (p \u3c 0.05) while the percentage of early apoptotic cells decreased (p \u3c 0.01) in cells transfected with the ATR plasmid containing the intact sORF. These findings have implications for the regulation of ATRs in physiological and pathological conditions and warrant investigation of sORFs in the 5\u27 leader sequence (5\u27LS) of other GPCRs