TGF-ß3 Induces Autophagic Activity by Increasing ROS Generation in a NOX4-Dependent Pathway.

Higher concentrations of reactive oxygen species (ROS) have been associated with epithelial cell damage, cell shedding, and airway hyperresponsiveness. Previous studies have indicated that transforming growth factor-beta (TGF-ß) mediates ROS production and NADPH oxidase (NOX) activity. In our previous study, we also observed that TGF-ß3 increases mucus secretion in airway epithelial cells in an autophagy-dependent fashion. Although it is well known that the relationship between ROS and autophagy is cell context-dependent, the exact mechanism of action remains unclear. The following study examined whether ROS act as upstream of autophagy activation in response to TGF-ß3 induction. Using an allergic inflammation mouse model induced by house dust mite (HDM), we observed elevated lung amounts of TGF-ß3 accompanied by increased ROS levels. And we found that ROS levels were elevated and NOX4 expression was increased in TGF-ß3-induced epithelial cells, while the lack of NOX4 in the epithelial cells could reduce ROS generation and autophagy-dependent MUC5AC expression treated with TGF-ß3. Furthermore, our studies demonstrated that the Smad2/3 pathway was involved in TGF-ß3-induced ROS generation by promoting NOX4 expression. The inhibition of ROS generation by N-Acetyl-L-cysteine (NAC) resulted in a decrease in mucus expression and autophagy activity in vivo as well as in vitro. Finally, TGF-ß3-neutralizing antibody significantly reduced the ROS generation, mucus expression, and autophagy activity and also decreased the phosphorylation of Smad2 and Smad3. Taken together, the obtained results revealed that persistent TGF-ß3 activation increased ROS levels in a NOX4-dependent pathway and subsequently induced autophagy as well as MUC5AC expression in the epithelial cells.

Development and validation of an LC/MS/MS method for simultaneous determination of shionone and epi-friedelinol in rat plasma for pharmacokinetic study after oral administration of Aster tataricus extract.

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated using spinasterol as the internal standard (IS) for the simultaneous determination of shionone and epi-friedelinol in rat plasma. Plasma samples were pretreated using liquid-liquid extraction with ethyl ether. Chromatographic separation was achieved on a C18 column (100 × 2.1 mm, 5 µm) with an isocratic elution consisting of acetonitrile-0.1% formic acid water (75:25, v/v) at a flow rate of 0.30 mL/min. Detection was performed under the selected reaction monitoring scan using an electrospray ionization in the positive ion mode. The mass transitions were as follows: m/z 427.4 → 95.1 for shionone, m/z 411.4 → 205.2 for epi-friedelinol and m/z 395.3 → 105.2 for IS. All calibration curves exhibited good linearity (r > 0.995) over the concentration range for both components. The intra- and inter-day precisions at three QC and lower limit of quantitation levels were both <10.21% in terms of relative standard deviation, and the accuracy ranged from -7.13 to 8.02% in terms of relative error. The extraction recoveries of the compounds ranged from 82.07 to 89.81%. The developed method was successfully applied to the pharmacokinetic study of shionone and epi-friedelinol after oral administration of Aster tataricus extract to rats. Copyright © 2015 John Wiley & Sons, Ltd.

Mutation spectrum of the CFTR gene in Taiwanese patients with congenital bilateral absence of the vas deferens.

BACKGROUND: Clinically affected cystic fibrosis (CF) patients present a spectrum of genital phenotypes ranging from normal fertility to moderately impaired spermatogenesis and congenital bilateral absence of vas deferens (CBAVD). Little is known about the CF incidence in the Taiwanese population. It has been shown that the CBAVD in men without clinical evidence of CF is associated with a high incidence of mutated CFTR (cystic fibrosis transmembrane conductance regulator) alleles. In order to understand the involvement of the CFTR gene in the aetiology of Asian/Taiwanese male infertility, we screened the entirety of the CFTR gene in 36 infertile males with CBAVD. METHODS: Temporal temperature gradient gel electrophoresis (TTGE) followed by direct DNA sequencing was used. RESULTS: Five mutations, p.V201M, p.N287K, c.-8G > C (125G > C), p.M469I and p.S895N, were found in five of the patients. p.N287K occurred in the first transmembrane-spanning domain, p.M469I in the first ATP-binding domain and p.S895N in the second transmembrane-spanning domain, were novel. In addition, seven homozygous and seven heterozygous 5T alleles in the intron 8 poly(T) tract were found. The overall frequency of CFTR mutant alleles in Taiwanese CBAVD males was 26 out of 72 = 36%. This finding was lower than the published frequency of CFTR mutations in other ethnic CBAVD patients (ranging from 50 to 74%). The frequency of p.M470V in Taiwanese CBAVD patients is not significantly different from that in the general population (P = 0.12). CONCLUSIONS: The results of this study add to the short list of Taiwanese/Asian CFTR mutations. Unlike Caucasian patients, the CFTR mutations cannot account for the majority of Taiwanese CBAVD. This is consistent with the low incidence of CF in the Asian/Taiwanese population. Furthermore, the mutation spectrum of CFTR in CBAVD patients does not overlap with the Caucasian CFTR mutation spectrum.

[Comparison of NP and MVP regimen in treatment of advanced non-small cell lung cancer].

BACKGROUND & OBJECTIVE: Chemotherapy is the major treatment for advanced non-small cell lung cancer (NSCLC). However, the efficacy is not satisfactory. From January 1996 to December 2000, two chemotherapy regimen [NP: vinorelbine(NVB) + cisplatin(DDP); MVP: mitomycin (MMC) + vindesine(VDS) + cisplatin] have been used to treat 110 advanced NSCLC patients. The response and major adverse reaction were analyzed and compared. METHODS: Forty-eight cases of advanced NSCLC (stage III-IV) patients were treated with NP (NVB: 25 mg/m2, d1, 8; DDP: 35 mg/m2, d1-3). The other 62 cases were treated with MVP regimen (MMC: 6 mg/m2, d1; VDS: 3 mg/m2, d1, 8; DDP: 30 mg/m2 d1-3). RESULTS: In NP group, the overall response rate was 50% (CR + PR = 24); medium response time was 5.5 months; medium survival time was 11 months. In MVP group, the overall response rate was 51.6% (CR + PR = 32), medium response time and survival time were 6.5 and 14.5 months, respectively. The major toxicities were myelosuppression and phlebitis in NP group, nausea/vomiting, myelosuppression in MVP group, respectively. CONCLUSION: NP and MVP regimen for advanced NSCLC have similar response rate (P > 0.05). Deep vein injection and improved infusion can be used to prevent phlebitis in NP regimen.