The relationship between the composition of SaPI1 transducing particles and those of helper phage 80␣ was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80␣ and SaPI1 virions, and all were encoded by 80␣. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.

A prototype electronic cigaret device and three formulations were evaluated in a 90-day rat inhalation study followed by a 42-day recovery period. Animals were randomly assigned to groups for exposure to low-, mid- and high-dose levels of aerosols composed of vehicle (glycerin and propylene glycol mixture); vehicle and 2.0% nicotine; or vehicle, 2.0% nicotine and flavor mixture. Daily targeted aerosol total particulate matter (TPM) doses of 3.2, 9.6 and 32.0 mg/kg/day were achieved by exposure to 1 mg/L aerosol for 16, 48 and 160 min, respectively. Pre-study evaluations included indirect ophthalmoscopy, virology and bacteriological screening. Body weights, clinical observations and food consumption were monitored weekly. Plasma nicotine and cotinine and carboxyhemoglobin levels were measured at days 28 and 90. After days 28, 56 and 90, lung function measurements were obtained. Biological endpoints after 90-day exposure and 42-day recovery period included clinical pathology, urinalysis, bronchoalveolar fluid (BALF) analysis, necropsy and histopathology. Treatment-related effects following 90 days of exposure included changes in body weight, food consumption and respiratory rate. Dose-related decreases in thymus and spleen weights, and increased BALF lactate dehydrogenase, total protein, alveolar macrophages, neutrophils and lung weights were observed. Histopathology evaluations revealed sporadic increases in nasal section 1–4 epithelial hyperplasia and vacuolization. Following the recovery period, effects in the nose and BALF were persistent while other effects were resolved. The no observed effect level based upon body weight decreases is considered to be the mid-dose level for each formulation, equivalent to a daily TPM exposure dose of approximately 9.6 mg/kg/day.

The chicken embryonic ␤-type globin gene, , is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated -globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zincfinger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pulldown assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive -globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active ␤ A -globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent -globin IntroductionMethylation of the 5-position of cytosine residues in DNA has an important role in the regulation of gene expression in higher eukaryotes. The first descriptions of an inverse correlation between DNA methylation and expression of a protein-coding eukaryotic gene were those made for the vertebrate globin genes of the chicken, rabbit, and human. [1][2][3][4] The tremendous interest in DNA methylation seen within the past decade stems from its important role in the silencing of tumor suppressor genes in cancer. 5,6 Despite the prediction of a widespread role for DNA methylation in gene regulation, 7 the expression of only a small number of vertebrate genes has been shown to be tissue-restricted or developmentally restricted by DNA methylation. 8 Studies showing tissue-restricted or developmentally restricted expression in nontransformed, primary cells are even more scarce and are limited to 4 main examples: the Gallus gallus -globin gene, 9,10 the Mus musculus interleukin-4 and interferon-␥ genes, 11 and the Xenopus laevis mesodermal genes. 12 Intense study has been directed toward elucidating the mechanisms through which DNA methylation represses transcription. The discovery of a family of proteins that specifically recognize methylated DNA, the methyl-CpG-binding proteins (MCBPs), 13,14 has led to the general observation of these proteins and their associated corepressor complexes as the mediators of DNA methylation-induced gene silencing in a variety of systems. [15][16][17][18][19] Biochemical studies have shown that the MCBPs are members of distinct and nonoverlapping transcriptional repression complexes. MBD1 was identified as a critical component of an S phase-specific complex that propagates the DNA methylation signal into a dimethylation of lysine 9 of histone H3 (H3-K9-Me 2 ) signal during DNA replication. 17 MBD2 is the methyl-CpG-binding component of the MeCP1 transcriptional repression complex. 20 MBD3 is a core component of the NuRD transcriptional repression complex that can be recruited by ...

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n = 18) or slow (n = 18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.

Secreted proteins, the secretome, can be isolated from biological fluids (e.g., blood) and are often responsible for the regulation of biological processes such as cell signaling, growth, and apoptosis. The identification of secreted proteins can lead to an understanding of disease mechanisms and they can serve as early candidate biomarkers of disease and exposure. However, it is time-consuming and costly to conduct in vivo interrogations of the human secretome. The purpose of this article is to provide a detailed description of a rapid in vitro technique for the analysis of differential protein secretion due to exposure to smoking-machine-generated cigarette smoke (CS) condensate (total particulate matter, TPM). Endothelial cells were exposed to CS-TPM, the supernatant was collected, and the secretome was elucidated by nano liquid chromatography coupled with high-resolution mass spectrometry. A total of 1,677 unique peptides were identified in the cell culture supernatants. Several proteins were differentially expressed following CS-TPM exposure that relate to several biological processes, such as metabolism, development, communication, response to stimulus, and response to stress.