Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

BACKGROUND: The functional role of nitric oxide (NO) and various nitric oxide synthase (NOS) isoforms in asthma remains unclear. OBJECTIVE: This study investigated the effects of ozone and ovalbumin (OVA) exposure on NOS isoforms. METHODS: The expression of inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) in lung tissue was measured. Enhanced pause (P(enh)) was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL) fluid were measured using a modified Griess reaction. RESULTS: The nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced P(enh )was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group. CONCLUSION: In mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

BACKGROUND: Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease. METHODS: We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy. RESULTS: The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks. CONCLUSION: These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR

Male Weaponry in a Fighting Cricket

Sexually selected male weaponry is widespread in nature. Despite being model systems for the study of male aggression in Western science and for cricket fights in Chinese culture, field crickets (Orthoptera, Gryllidae, Gryllinae) are not known to possess sexually dimorphic weaponry. In a wild population of the fall field cricket, Gryllus pennsylvanicus, we report sexual dimorphism in head size as well as the size of mouthparts, both of which are used when aggressive contests between males escalate to physical combat. Male G. pennsylvanicus have larger heads, maxillae and mandibles than females when controlling for pronotum length. We conducted two experiments to test the hypothesis that relatively larger weaponry conveys an advantage to males in aggressive contests. Pairs of males were selected for differences in head size and consequently were different in the size of maxillae and mandibles. In the first experiment, males were closely matched for body size (pronotum length), and in the second, they were matched for body mass. Males with proportionately larger weaponry won more fights and increasing differences in weaponry size between males increased the fighting success of the male with the larger weaponry. This was particularly true when contests escalated to grappling, the most intense level of aggression. However, neither contest duration nor intensity was related to weaponry size as predicted by models of contest settlement. These results are the first evidence that the size of the head capsule and mouthparts are under positive selection via male-male competition in field crickets, and validate 800-year-old Chinese traditional knowledge

Adjuvant concurrent chemoradiation therapy (CCRT) alone versus CCRT followed by adjuvant chemotherapy: Which is better in patients with radically resected extrahepatic biliary tract cancer?: a non-randomized, single center study

<p>Abstract</p> <p>Background</p> <p>There is currently no standard adjuvant therapy for patients with curatively resected extrahepatic biliary tract cancer (EHBTC). The aim of this study was to analyze the clinical features and outcomes between patients undergoing adjuvant concurrent chemoradiation therapy (CCRT) alone and those undergoing CCRT followed by adjuvant chemotherapy after curative resection.</p> <p>Methods</p> <p>We included 120 patients with EHBTC who underwent radical resection and then received adjuvant CCRT with or without further adjuvant chemotherapy between 2000 and 2006 at Seoul National University Hospital.</p> <p>Results</p> <p>Out of 120 patients, 30 received CCRT alone, and 90 received CCRT followed by adjuvant chemotherapy. Baseline characteristics were comparable between the two groups. Three-year disease-free survival (DFS) rates for CCRT alone and CCRT followed by adjuvant chemotherapy were 26.6% and 45.2% (p = 0.04), respectively, and 3-year overall survival (OS) rates were 30.8% and 62.6% (p < 0.01), respectively. CCRT followed by adjuvant chemotherapy showed longer survival than did CCRT alone, especially in R1 resection (microscopically positive margins) or negative lymph node.</p> <p>Conclusion</p> <p>Adjuvant CCRT followed by adjuvant chemotherapy prolonged DFS and OS, compared with CCRT alone in patients with curatively resected EHBTC. Adjuvant chemotherapy deserves to consider after adjuvant CCRT. In the future, a randomized prospective study will be needed, with the objective of investigating the role of adjuvant chemotherapy.</p

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

Background: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. in our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.Methods: the beta 1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and beta 1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.Results: Differential association among Timp1, CD63 and beta 1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. in human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.Conclusions: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and beta 1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. in addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilLudwig Inst Canc Res, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/12306-1FAPESP: 2010/18715-8CAPES: 2867/10Web of Scienc

Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences

<p>Abstract</p> <p>Background</p> <p>Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated <it>AAVS1 </it>(also known as <it>MBS85</it>). Integration at <it>AAVS1 </it>requires the AAV2 replication (Rep) proteins and a DNA sequence within <it>AAVS1 </it>containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or <it>trs</it>). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced <it>trs</it>, but the sequences differ from those in <it>AAVS1</it>. These ITR sequences are required for replication and packaging.</p> <p>Results</p> <p>In this study we demonstrate that the <it>AAVS1 </it>RRS and <it>trs </it>can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of <it>AAVS1 </it>DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at <it>AAVS1</it>, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to <it>AAVS1 </it>sequences.</p> <p>Conclusions</p> <p>The ability of these <it>AAVS1 </it>sequences to substitute for the AAV2 RRS and <it>trs </it>provides indirect evidence that the stable secondary structure encompassing the <it>trs </it>is part of the AAV2 packaging signal.</p

Combined Inactivation of MYC and K-Ras Oncogenes Reverses Tumorigenesis in Lung Adenocarcinomas and Lymphomas

Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.To examine how the MYC and K-ras(G12D) oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D) to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D)- or MYC/K-ras(G12D)-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D)-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D) resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D) in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D) signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D) but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D). Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas