Modulation of the epidermal growth factor receptor by brain‐derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37°C with bovine brain‐derived growth factor (BDGF) decreased the cell surface 125I‐EGF binding activity of these cells by 70–80%. This down‐modulation of the EOF receptor by BDGF was time, temperature, and dose dependent Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high‐affinity EGF receptors. The BDGF‐induced down‐modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down‐modulated the EGF receptor in phorbol myristic acetate (PMA)‐pretreated cells, as well as in control cells. Furthermore, PMA‐pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA‐pretreated cells. This BDGF‐induced down‐modulation of the EGF receptor in PMA‐desensitized cells suggests that BDGF down‐regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down‐modulation induced either by BDGF or by platelet‐derived growth factor (PDGF) but had no effect on the PMA‐induced down‐modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF‐induced down‐modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down‐modulation of the EGF receptor by increasing the internalization of cell surface high‐affinity receptors and that the internalization process may not be required for down‐modulation induced by PMA

Cellular growth inhibition by TGF-β1 involves IRS proteins

AbstractIn Mv1Lu cells, insulin partially reverses transforming growth factor-β1 (TGF-β1) growth inhibition in the presence of α5β1 integrin antagonists. TGF-β1 appears to induce phosphorylation of IRS-2 in these cells; this is inhibited by a TGF-β antagonist known to reverse TGF-β growth inhibition. Stable transfection of 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by TGF-β1) with IRS-1 or IRS-2 cDNA confers sensitivity to growth inhibition by TGF-β1; this IRS-mediated growth inhibition can be partially reversed by insulin in 32D cells stably expressing IRS-2 and the insulin receptor (IR). These results suggest that growth inhibition by TGF-β1 involves IRS proteins

Inhibitors of clathrin-dependent endocytosis enhance TGFβ signaling and responses

Clathrin-dependent endocytosis is believed to be involved in TGFβ-stimulated cellular responses, but the subcellular locus at which TGFβ induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFβ and promote colocalization and accumulation of TβR-I and SARA at the plasma membrane. These inhibitors enhance TGFβ-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFβ. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFβ responsiveness caused by the hypercholesterolemia, presumably acting through its effect on TGFβ endocytosis and signaling in vascular cells

Incidence of and Factors Associated with False Positives in Laboratory Diagnosis of Norovirus Infection by Amplification of the RNA-Dependent RNA Polymerase Gene

<div><p>Background</p><p>Conventional reverse transcription-polymerase chain reaction (RT-PCR) amplification of the RNA-dependent RNA polymerase (RdRp) gene remains a used method for the rapid detection of norovirus (NV) in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated.</p><p>Methods/Principal Findings</p><p>After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6%) samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91–97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17–37.92, <i>P</i> = .003) and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21–24.73, <i>P</i> = .027) as significant and independent factors associated with false positives.</p><p>Conclusion</p><p>Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.</p></div