Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype

PDGF-C Mediates the Angiogenic and Tumorigenic Properties of Fibroblasts Associated with Tumors Refractory to Anti-VEGF Treatment

SummaryTumor- or cancer-associated fibroblasts (TAFs or CAFs) from different tumors exhibit distinct angiogenic and tumorigenic properties. Unlike normal skin fibroblasts or TAFs from TIB6 tumors that are sensitive to anti-VEGF treatment (TAF-TIB6), TAFs from resistant EL4 tumors (TAF-EL4) can stimulate TIB6 tumor growth even when VEGF is inhibited. We show that platelet-derived growth factor C (PDGF-C) is upregulated in TAFs from resistant tumors. PDGF-C-neutralizing antibodies blocked the angiogenesis induced by such TAFs in vivo, slowed the growth of EL4 and admixture (TAF-EL4 + TIB6) tumors, and exhibited additive effects with anti-VEGF-A antibodies. Hence, our data reveal an additional mechanism for TAF-mediated tumorigenesis and suggest that some tumors may overcome inhibition of VEGF-mediated angiogenesis through upregulation of PDGF-C

Model Pembelajaran Asam Basa Berbasis Scs (Science Process Skills) Melalui Kegiatan Laboratorium Sebagai Wahana Pendidikan Sains Siswa Mts

Tujuan penelitian ini adalah untuk memperkenal- kan dan mengetahui efektivitas Model Pembelajaran SCS ( Science Process Skills ) melalui kegiatan Laboratorium Sebagai Wahana Pendidikan Sains yang cocok bagi siswa MTs agar meningkatkan: penguasaan konsep kimia, ke- mampuan berpikir kreatif, dan keterampilan sains siswa. Metode yang digunakan adalah metode penelitian kelas, dan difokuskan pada pokok bahasan asam basa. Penelitian ini dilakukan di salah satu MTs Negeri di kota Semarang dengan subyek sebanyak 40 siswa kelas III. Instrumen yang digunakan dalam penelitian ini meliputi model pembelaja- ran, soal-soal tes, pedoman wawancara, pedoman obser- vasi dan angket, sedang LKS digunakan pada saat kegiatan laboratorium. Dalam model pembelajaran dikembangkan empat jenis konsep yaitu konsep kongkret, konsep yang menyatakan sifat, konsep yang melibatkan penggambaran simbol, dan konsep berdasarkan prinsip. Model pembela- jaran ini dapat meningkatkan pemahaman konsep pada setiap kelompok kemampuan siswa, mengembangkan ke- mampuan berpikir kreatif dengan hasil tertinggi pada as- pek membangun konsep di atas pengetahuan yang telah ada pada diri siswa dan terendah pada aspek memilih hal- hal yang mungkin tidak relevan, serta keterampilan sains mengatasi kurangnya waktu pembelajaran, bagian-bagian pembelajaran tertentu dapat dilaksanakan di luar jam ke- las

Stromal Cell-Derived Factor-1/CXCL12 Contributes to MMTV-Wnt1 Tumor Growth Involving Gr1+CD11b+ Cells

BACKGROUND: Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1(+) myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1(+) cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1(+) cells. CONCLUSIONS/SIGNIFICANCE: We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1(+) myeloid cells to facilitate growth and/or angiogenesis

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

High-resolution analysis of protein, and DNA conformational changes during DNA polymerization, established relationships between the enzymatic function and conformational dynamics of individual domains for a DNA polymerase

Clinical Characteristics of 26 Human Cases of Highly Pathogenic Avian Influenza A (H5N1) Virus Infection in China

BACKGROUND: While human cases of highly pathogenic avian influenza A (H5N1) virus infection continue to increase globally, available clinical data on H5N1 cases are limited. We conducted a retrospective study of 26 confirmed human H5N1 cases identified through surveillance in China from October 2005 through April 2008. METHODOLOGY/PRINCIPAL FINDINGS: Data were collected from hospital medical records of H5N1 cases and analyzed. The median age was 29 years (range 6-62) and 58% were female. Many H5N1 cases reported fever (92%) and cough (58%) at illness onset, and had lower respiratory findings of tachypnea and dyspnea at admission. All cases progressed rapidly to bilateral pneumonia. Clinical complications included acute respiratory distress syndrome (ARDS, 81%), cardiac failure (50%), elevated aminotransaminases (43%), and renal dysfunction (17%). Fatal cases had a lower median nadir platelet count (64.5 x 10(9) cells/L vs 93.0 x 10(9) cells/L, p = 0.02), higher median peak lactic dehydrogenase (LDH) level (1982.5 U/L vs 1230.0 U/L, p = 0.001), higher percentage of ARDS (94% [n = 16] vs 56% [n = 5], p = 0.034) and more frequent cardiac failure (71% [n = 12] vs 11% [n = 1], p = 0.011) than nonfatal cases. A higher proportion of patients who received antiviral drugs survived compared to untreated (67% [8/12] vs 7% [1/14], p = 0.003). CONCLUSIONS/SIGNIFICANCE: The clinical course of Chinese H5N1 cases is characterized by fever and cough initially, with rapid progression to lower respiratory disease. Decreased platelet count, elevated LDH level, ARDS and cardiac failure were associated with fatal outcomes. Clinical management of H5N1 cases should be standardized in China to include early antiviral treatment for suspected H5N1 cases

Evidence for Pro-angiogenic Functions of VEGF-Ax.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1

Evidence for Pro-angiogenic Functions of VEGF-Ax.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1

Transmembrane transforming growth factor-α tethers to the PDZ domain-containing, Golgi membrane-associated protein p59/GRASP55

Transforming growth factor-α (TGF-α) and related proteins represent a family of transmembrane growth factors with representatives in flies and worms. Little is known about the transport of TGF-α and other transmembrane growth factors to the cell surface and its regulation. p59 was purified as a cytoplasmic protein, which at endogenous levels associates with transmembrane TGF-α. cDNA cloning of p59 revealed a 452 amino acid sequence with two PDZ domains. p59 is myristoylated and palmitoylated, and associates with the Golgi system, where it co-localizes with TGF-α. Its first PDZ domain interacts with the C-terminus of transmembrane TGF-α and select transmembrane proteins. p59 is the human homolog of GRASP55, which is structurally related to GRASP65. GRASP55 and GRASP65 have been shown to play a role in stacking of the Golgi cisternae in vitro. C-terminal mutations of transmembrane TGF-α, which decrease or abolish the interaction with p59, also strongly impair cell surface expression of TGF-α. Our observations suggest a role for membrane tethering of p59/GRASP55 to select transmembrane proteins, including TGF-α, in maturation and transport to the cell surface