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Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10-24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10-3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10-3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10-3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10-3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10-4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10-5). KC corneas also had increased mtDNA damage (P = 3.63×10-10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.

Vitamin E (VE) can effectively prevent occurrence of lung cancer caused by passive smoking in mice. However, whether VE prevents smoking-induced cytotoxicity remains unclear. In this study, a primary culture of embryonic lung cells (ELCs) was used to observe the cytotoxic effects of cigarette smoke extract (CSE), including its influence on cell survival, cell cycle, apoptosis, and DNA damage, and also to examine the effects of VE intervention on CSE-induced cytotoxicity. Our results showed that CSE could significantly inhibit the survival of ELCs with dose- and time-dependent effects. Furthermore, CSE clearly disturbed the cell cycle of ELCs by decreasing the proportion of cells at the S and G2/M phases and increasing the proportion of cells at the G0/G1 phase. CSE promoted cell apoptosis, with the highest apoptosis rate reaching more than 40%. CSE also significantly caused DNA damage of ELCs. VE supplementation could evidently inhibit or reverse the cytotoxic effects of CSE in a dose- and time-dependent manner. The mechanism of CSE effects on ELCs and that of VE intervention might involve the mitochondrial pathway of cytochrome c-mediated caspase activation. Our study validate that VE plays a clearly protective effect against CSE-induced cytotoxicity in mouse embryonic lung cells.

Objective: Grb2-associated binder 2 (Gab2), a member of the family of Gab scaffolding adaptors, transmits and amplifies the signals from receptor tyrosine kinases. A recent study demonstrated that Gab2 was over-expressed in breast cancers and metastatic melanomas, and Gab2 was an oncogenic protein. However, the roles of Gab2 in lung cancers are largely unknown.Method: In this study, to investigate whether Gab2 expression could be a characteristic of lung cancers, we analyzed the expression of Gab2 in 88 lung frozen tissue samples and 122 paraffin-embedded tissue specimens, using quantitative real-time-PCR, immunohistochemistry and western blot.Results: We found that the positive expression rate of Gab2 in the tumor tissues, as detected by immunohistochemistry, 62.5% in squamous cell cancers, 51.35% in adenocarcinomas, and 75% in other types of lung cancers, was significantly higher than that (12%) in normal lung tissues. The mRNA expression detected by quantitative real-time-PCR and protein expression detected by western blotting in different groups were consistent with the immunohistochemical results.Conclusion: Our data indicate that Gab2 is over-expressed in malignant lung tissues compared with that in normal lung tissues, and suggest that Gab2 expression may play a role in lung cancer development.

OBJECTIVE To explore the relationship between CAR and the development of human lung cancer, as well as to provide the basis for the clinical treatment of lung cancer using an adenovirus vector-based gene therapy. METHODS CAR expression was assessed immunohisto-chemically in tumoral, paraneoplastic and normal samples from 112 lung cancer patients. At the same time, the mRNA and protein expression of CAR in 32 cases were determined by RT-PCR and Western blot. The relationship between CAR expression and clinicopathologic parameters was statistically analyzed. RESULTS There was no expression of CAR in normal lung tissue but a little in paraneoplastic tissue. The positive rate was 43% in squamous cell carcinoma, and 70% in adenocarcinoma. Both were much significantly higher than that in paraneoplastic tissue. The CAR expression level in adenocarcinoma was higher than that in squamous cell cancer. mRNA expression by RT-PCR and protein expression by Western blot were consistent with immunohistochemistry results. CONCLUSION CAR is overexpressed in human lung cancer, especially in adenocarcinoma. This data offer the reliable basis for adenovirus-mediated gene therapy of lung cancer; more important, CAR may take part in the formation or development of lung cancer; this may be exploitable for the development of antibody-directed therapy in human lung cancer.

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.