Rationale: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. Objectives: To investigate the role of extracellular histones in COPD disease progression. Methods: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. Measurements and Main Results: A striking finding was a COPDspecific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca 21 homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. Conclusions: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.Keywords: chronic obstructive pulmonary disease; histone H3.3; acetylation; cytotoxicity; proteomicsThe prevalence of chronic obstructive pulmonary disease (COPD) is increasing in industrialized countries (1, 2). COPD is the third leading cause of death worldwide (3). In 2011, the CDC estimated that 15 million adults in the United States had COPD, which contributed to $49 billion in direct and indirect healthcare costs (4, 5). Disease management can relieve symptoms and prolong life, although there are no treatments to stop the disease progression, which ultimately results in death.Although the molecular pathophysiology that underlies COPD has not been established, it is known that the expression of proinflammatory molecules, lung cell death, and tissue remodeling play critical roles (2, 6-8). Shifts in transcription factor activation and epigenetic markers, such as altered histone acetylation, are associated with changes in signaling proteins upstream of regulatory genes and in the assembly of nuclear chromatin complexes (9, 10). Core histones H2A, H2B, H3, H4 and their variants are key components of nuclear chromatin, the scaffolding that controls interactions between DNA with transcription factors and RNA polymerase, thereby regulating gene expression (11). Post-translational modification of histones causes chromatin ...

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.

Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 g of R-(؉) nicotine kg ؊1 h ؊1 intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 g kg ؊1 h ؊1 into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet.

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Leptin is overexpressed in human breast tumors and is produced by breast cancer cells in response to obesity‐related stimuli. The leptin promoter polymorphism Lep‐2548G/A can be associated with increased leptin secretion by adipocytes and elevated cancer risk. However, molecular mechanisms underlying the link between Lep‐2548G/A and breast cancer have never been addressed. Lep‐2548G/A is proximal to a binding site for the transcriptional factor Sp1. Furthermore nucleolin, a transcriptional repressor, can bind Sp1 or its consensus site. Consequently, we focused on the impact of Lep‐2548G/A on Sp1‐ and nucleolin‐dependent leptin transcription in breast cancer cells. The Lep‐2548G/A was identified in a homozygous conformation in BT‐474 and SK‐BR‐3 breast cancer cells, in a heterozygous conformation in MDA‐MB‐231 cells, and a wild‐type Lep‐2548G/G sequence was present in MCF‐7 and ZR‐75‐1 cells. The occurrence of Lep‐2548A/A and Lep‐2548G/A coincided with high and intermediate leptin mRNA expression, respectively, while cells containing Lep‐2548G/G expressed low leptin mRNA levels. We demonstrated that the existence of Lep‐2548G/A improved efficient recruitment of Sp1 to DNA under insulin treatment, while Sp1 loading on DNA containing Lep‐2548G/G was not insulin‐dependent. In contrast, nucleolin binding to Lep‐2548G/A was downregulated in response to insulin, while it was not regulated on Lep‐2548G/G. The presence of Lep‐2548G/A was studied in breast cancer epithelial cells by IHC and LCM. Interestingly, all 14 tumors expressing high leptin levels contained Lep‐2548A/A. In conclusion, the occurrence of Lep‐2548G/A can enhance leptin expression in breast cancer cells via Sp1‐ and nucleolin‐dependent mechanisms and possibly contribute to intratumoral leptin overexpression. © 2009 UICC

The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.

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