Airway tissue factor-dependent coagulation activity in response to sulfur mustard analog 2-chloroethyl ethyl sulfide.

Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.

The reversibility of mitotic exit in vertebrate cells.

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.

Impact of SOD-Mimetic Manganoporphyrins on Spin Trapping of Superoxide and Related Artifacts.

The superoxide dismutase (SOD)-mimetic effectiveness of [meso-tetrakis (R)porphyrinato]manganese with R = 1,3-di-N-ethylimidazolium-2-yl (Mn-TDEIP), 1,3-di-N-methylimidazolium-2-yl (Mn-TDMIP), 1,3-di-N-propylimidazolium-2-yl (Mn-TDPIP), N-ethyl-2-pyridyl (Mn-T2EPyP), 4-sulphonatophenyl (Mn-TSP), 1-methyl-4-pyridyl (Mn-T4PyP), 4-carboxyphenyl (Mn-TBAP), and ß-octabromomeso-tetrakis(4-carboxyphenyl porphyrinato)manganese (MnBr8TBAP) was compared with Cu, Zn SOD. Superoxide generated by reaction of xanthine oxidase with hypoxanthine was trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), forming BMPO-OOH, which was monitored by electron paramagnetic resonance. Manganoporphyrins with redox potentials ranging from -0.190 to 0.346 V relative to the standard hydrogen electrode were selected for this study. With 0.1 µM manganoporphyrins and 20 mM BMPO, the effectiveness of the manganoporphyrins in inhibiting formation of BMPO-OOH increases in the order Mn-TSP < Mn-TBAP < MnBr8TBAP < Mn-T4PyP < Mn-T2EPyP < Mn-TDEIP ~ Mn-TDMIP ~ Mn-TDPIP ~ Cu, Zn SOD. However, at higher concentrations of manganoporphyrin and BMPO, a BMPO-OH signal was observed. The formation of BMPO-OH was not inhibited by catalase or dimethylsulfoxide, which demonstrated that it was not produced from hydroxyl radical. The artifactual formation of BMPO-OH is attributed to oxidation of the water adduct of BMPO by the manganoporphyrins or decomposition of BMPO-OOH. Although spin trapping is an effective method for evaluating SOD-mimetic efficacy, caution must be exercised to ensure that artifact signals are not interpreted improperly.

Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.

RATIONALE: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown. OBJECTIVES: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken. METHODS: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots. MEASUREMENTS AND MAIN RESULTS: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha. CONCLUSIONS: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.

Targeting of a novel fusion protein containing methioninase to the urokinase receptor to inhibit breast cancer cell migration and proliferation.

It has been shown that methionine depletion inhibits tumor cell growth and reduces tumor cell survival. A novel fusion protein targeted specifically to tumor cells was developed. The fusion protein contained two components: the amino terminal fragment of human urokinase (amino acids 1-49) that binds to the urokinase receptor protein expressed on the surface of invasive cancer cells, and the enzyme L-methioninase (containing 398 amino acids) which depletes methionine and arrests the growth of methionine-dependent tumors. The influence of the fusion protein on the growth and motility of human breast cancer cells was examined using a culture wounding assay. It was determined that MCF-7 breast cancer cells, used in this study, were methionine-dependent and that the fusion protein bound specifically to urokinase receptors of the surface of the cancer cells. Further treatment of the cancer cells with fusion protein over the concentration range 10(-8) to 10(-6) M produced a dose-dependent inhibition of both the migration and proliferation index of MCF-7 cells in the culture wounding assay over a period of 1 to 3 days. The results of this study suggest that this novel fusion protein may serve as a prototype for specific targeting of methioninase and perhaps other cytotoxic agents to cancer cells.

Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery.

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.