Chain transfer to polymer in free-radical bulk and emulsion polymerization of vinyl acetate has been studied using 13 C NMR spectroscopy. The poly(vinyl acetate) (PVAc) spectra have been fully interpreted in terms of structural features arising from (i) normal and inverted repeat unit placements, (ii) chain transfer to polymer, and (iii) in the case of emulsion polymerization, hydrolysis of acetate side groups. Although evidence was obtained for chain transfer to polymer via H-abstraction from both backbone tertiary C-H bonds and methyl side groups, the latter make the dominant contribution. The spectral assignments facilitated calculation of the mole percent branches and the mole percent of inverted repeat units in the PVAc samples produced, the latter quantity taking values of 1.44 ( 0.24 mol % independent of conversion in the emulsion polymerizations. The absence of an initiator effect in the bulk polymerizations shows that the propagating PVAc chain radicals are the principal hydrogen atom abstractors. In both bulk and emulsion polymerization, the mole percent branches increased steadily with overall conversion. However, because the emulsion polymerizations proceed almost exclusively within the latex particles and at high instantaneous conversions, the levels of branching in the PVAc produced (final levels ) 0.61-0.75 mol %) were much higher than in the PVAc produced by bulk polymerization (final levels ) 0.13-0.23 mol %). Increasing the temperature of emulsion polymerization from 60 to 70 °C led to an increase in the mole percent branches but had no measurable effect on the mole percent of inverted repeat units.

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput. Molecular & Cellular Proteomics 9:1400 -1410, 2010.Oxidation of cysteine residues plays a critical role in modifying the structure and function of many proteins. Although cysteine oxidation is a tightly regulated biological process, nonenzymatic processes can contribute substantially to its levels, such as during oxidative stress. Regulatory oxidation states such as disulfide bonding and S-nitrosylation are readily modulated (1) and play an essential role in many physiological processes, including cell cycle, growth, death, and differentiation (2). In contrast, prolonged accumulation of reactive oxygen species is associated with many pathological conditions and leads to stable overoxidized states (sulfinic and sulfonic acid) that may disrupt redox regulation and protein function (3) and, in most cases, are thought to be nonregenerative.Assays capable of comprehensively assessing the dynamic changes in site-specific oxidation states are especially critical to understanding the contribution of redox status to many diseases. Numerous redox-sensitive proteins, including essential cellular regulators such as p53, have been described previously (for review, see ref. 4). However, technical factors have hampered the identification of specific site(s) of modification and characterization of their redox status in cells. Sitedirected mutagenesis is often employed to determine whether specific cysteines have redox-regulated functional roles (1), but this approach provides no information on the oxidation status of the endogenous protein. In addition, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants (5) and regulators of various antioxidant enzymes (6). Thiol pKa (7), solvent accessibility, and ...

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We have previously demonstrated that oestrogen receptor alpha (ERalpha) modulates epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signalling efficiency in a tamoxifen-resistant MCF-7 breast cancer cell line (Tam-R). In the present study we have investigated whether this cross-talk between EGFR/MAPK and ERalpha signalling pathways is bidirectional by examining the effects of EGFR/MAPK activity on ER functionality in the same cell line. Elevated expression levels of phosphorylated serine 118 (S118) ERalpha were observed in the Tam-R compared to the parental wild type MCF-7 cell line (WT-MCF-7) under basal growth conditions. Phosphorylation of ERalpha at S118 was regulated by the EGFR/MAPK pathway in Tam-R cells being increased in response to amphiregulin (AR) and inhibited by the selective EGFR tyrosine kinase inhibitor, gefitinib and the MEK1/2 inhibitor, PD184352. Recruitment of the co-activators p68 RNA helicase and SRC1 to ERalpha, oestrogen response element (ERE) activity and Tam-R cell growth were similarly EGFR/MAPK-regulated. Chromatin immunoprecipitation (ChIP) studies revealed that in Tam-R cells the ERalpha assembled on the AR gene promoter and this was associated with elevated basal expression of AR mRNA. Furthermore, AR mRNA expression was under the regulation of the EGFR/MAPK and ERalpha signalling pathways. Neutralising antibodies to AR inhibited EGFR/ERK1/2 activity, reduced S118 ERalpha phosphorylation and reduced AR mRNA expression in TAM-R cells. These findings suggest that ERalpha function in Tam-R cells is maintained as a consequence of EGFR/MAPK-mediated phosphorylation at serine residue 118 resulting in the generation of a self-propogating autocrine growth-regulatory loop through the ERalpha-mediated production of AR.