Molecular dynamics study of enhanced autophosphorylation by S904F mutation of the RET kinase domain.

The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant.

Curcumin analog, GO-Y078, induces HO-1 transactivation-mediated apoptotic cell death of oral cancer cells by triggering MAPK pathways and AP-1 DNA-binding activity.

BACKGROUND: GO-Y078, a new synthetic analogue of curcumin (CUR), has higher oral bioavailability and anticancer activity than CUR, but the oncostatic effect of GO-Y078 on oral squamous cell carcinoma (OSCC) is largely unknown. RESEARCH DESIGN AND METHODS: In the present study, we examined the oncostatic properties and possible mechanisms of GO-Y078 on human SCC-9 and HSC-3 OSCC cells. RESULTS: Our results indicated that GO-Y078 showed a cytostatic effect against OSCC cells, and this antiproliferative phenomenon stemmed from a mechanism involving multiple levels of cooperation, including cell-cycle G2/M arrest and apoptosis induction. Mechanistically, GO-Y078 treatment induced caspase-mediated apoptosis via upregulating two apoptosis-modulating proteins, SMAC/DIABLO and heme oxygenase (HO)-1. GO-Y078 transcriptionally induced upregulation of the HO-1 gene by increasing the AP-1 DNA-binding activity, which was initiated by activation of the p38 /JNK1/2 pathways. In the clinic, patients with head and neck cancers expressed lower HO-1 and SMAC/DIABLO levels in primary cancer tissues compared to normal tissues. Clinical datasets also revealed that patients with head and neck cancers expressing high HO-1 had afavorable prognosis. CONCLUSIONS: Our results provide new insights into the role of GO-Y078-induced molecular regulation in suppressing OSCC growth and suggest that GO-Y078 has potential therapeutic applications for OSCC.

Determination of activity of denitrifying enzyme in soil samples by a headspace gas chromatographic technique.

This paper reported a headspace gas chromatography (HS-GC) for the determination of denitrifying enzyme activity in soil samples. It was based on measuring the NO2 signal in a set of closed/air-free vials containing soil samples that incubated at the designated conditions. The results showed that the method has a good measurement precision (RSD < 5.0%) and reasonable accuracy for determining the denitrifying enzyme activity in soil samples. It is simple and efficient, and very suitable to be used in batch sample analysis.

Tai Chi Chuan Exercise Improves Lung Function and Asthma Control through Immune Regulation in Childhood Asthma.

BACKGROUND: Tai Chi Chuan (TCC) is an exercise of low to moderate intensity with key features of mindfulness, structural alignment, and flexibility to relax the body and mind in adults. Our previous study showed that TCC could improve the quality of life (QoL), pulmonary function, and fractional exhaled nitric oxide in asthmatic children. We further investigated whether the benefits induced by TCC were associated with immune regulation. METHOD: Six- to twelve-year-old children diagnosed with mild to severe persistent asthma for at least one year according to the Global Initiative for Asthma guidelines were enrolled from a tertiary pediatric allergy center in Taiwan. Asthmatic children were divided into two groups based on their choice: (1) the TCC group had a 60-minute TCC exercise session once weekly led by an instructor and (2) the control group kept their original activity levels. All other exercises were encouraged as usual. Pulmonary function tests, laboratory tests, standardized pediatric asthma QoL questionnaire (PAQLQ(S)), and childhood asthma control test (C-ACT) were performed before and after the TCC program (12 weeks). Data on medications and exacerbations were collected from medical records. RESULTS: There were no differences between the TCC (n = 25) and control (n = 15) groups at baseline, except that the C-ACT showed significantly lower results in the TCC group (p=0.045). After 12 weeks, the number of leukocytes (p=0.041) and eosinophils (p=0.022) decreased, while regulatory T cells increased significantly (p=0.008) only in the TCC group. Lung functions (FEV1 and PEFR) were significantly improved in both the TCC (p < 0.001) and control (p=0.045 and 0.019, respectively) groups, while the PAQLQ(S) and C-ACT (p < 0.001) showed improvement only in the TCC group. Moreover, compared to the control group, the exacerbations within 12 weeks after the study were significantly decreased in the TCC group (p=0.031). After multiple regression by a conditional forward method, the factors that were significantly associated with exacerbation within 12 weeks after study is the practice of TCC and exacerbation within 24 weeks before study (p=0.013 and 0.015, respectively) after adjusting for age, sex, asthma severity, PEF, FEV1, C-ACT, PAQLQ(S), and medication score at baseline. CONCLUSION: TCC exercise may improve pulmonary functions, asthma control, and QoL and prevent exacerbations in asthmatic children through immune regulation. Further research on detailed mechanisms is mandated.

Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species.

Amoxicillin is a commonly prescribed drug for anti- bacterial infection. In this study, we are interested in the effect of the drug on the cellular DNA integrity. Amoxicillin was added to the human or hamster cells in culture, and the DNA lesions induced by the drug were assessed by a comet assay with nuclear extract incubation (Wang et al., 2005 Anal Biochem 337: 70-75). Amoxicillin at 5mM rapidly induced DNA lesions in human AGS cells. The level of DNA lesions attained a maximum at about 1h, and then declined steadily and reached almost the basal level at 6h following the drug treatment. Similar induction pattern of DNA lesions was found with amoxicillin-related antibiotics such as ampicillin but not with the unrelated antibiotics such as kanamycin. For studying the repair kinetics, the cells were treated with amoxicillin for only 1h and continued culture in the absence of the drug for a certain period of time before subsequent analysis. Repair of the amoxicillin-induced DNA lesions was essentially completed within 4h. Such repair may not involve nucleotide excision repair (NER) pathway because the repair was completed with similar kinetics in both NER proficient Chinese hamster CHO-K1 cells and its isogenic NER deficient UV24 cells. Instead, the repair may involve base excision repair (BER) pathway because immunodepletion of OGG1/2, glycosylases involved in BER rendered the nuclear extract unable to excise DNA lesions induced by amoxicillin in the modified comet assay. Furthermore, amoxicillin induced intracellular reactive oxygen species (ROS) at the tempo similar to that of DNA lesions induction. Thus, we hypothesize that amoxicillin causes oxidative DNA damage in mammalian cells via ROS.

Tai-Chi-Chuan Exercise Improves Pulmonary Function and Decreases Exhaled Nitric Oxide Level in Both Asthmatic and Nonasthmatic Children and Improves Quality of Life in Children with Asthma.

Tai-Chi-Chuan (TCC) is an exercise of low-to-moderate intensity which is suitable for asthmatic patients. The aim of our study is to investigate improvements of the lung function, airway inflammation, and quality of life of asthmatic children after TCC. Participants included sixty-one elementary school students and they were divided into asthmatic (n = 29) and nonasthmatic (n = 32) groups by the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Among them, 20 asthmatic and 18 nonasthmatic children volunteered to participate in a 60-minute TCC exercise weekly for 12 weeks. Baseline and postintervention assessments included forced expiratory volume in one second (FEV1), forced vital capacity (FVC), peak expiratory flow rate (PEFR), fractional exhaled nitric oxide (FeNO) level, and Standardised Pediatric Asthma Quality of Life Questionnaire (PAQLQ(S)). After intervention, the level of FeNO decreased significantly; PEFR and the FEV1/FVC also improved significantly in both asthmatic group and nonasthmatic group after TCC. The asthmatic children also had improved quality of life after TCC. The results indicated that TCC could improve the pulmonary function and decrease airway inflammation in both children with mild asthma and those without asthma. It also improves quality of life in mild asthmatic children. Nevertheless, further studies are required to determine the effect of TCC on children with moderate-to-severe asthma.

Is the oxidative DNA damage level of human lymphocyte correlated with the antioxidant capacity of serum or the base excision repair activity of lymphocyte?

A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER) activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP) or Hepatitis B virus (HBV) tend to give higher levels of oxidative DNA damage (P < 0.05). Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P < 0.05). Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

HLA class II variants in Chinese breast cancer patients.

Alterations of human leukocyte antigen (HLA) class II molecules are relevant to the development of breast cancer and metastatic progression. However, the role of HLA class II polymorphisms in the pathogenesis and progression of breast cancer is unclear. This study aimed to investigate the association between HLA class II variants and breast cancer susceptibility and prognosis in a Chinese population. Sixteen variants in HLA class II were detected with the Sequenom MassArray® iPLEX System in 216 breast cancer patients and 216 healthy controls. An association analysis based on unconditional logistic regression was carried out to determine the odds ratio (OR) and 95% confidence interval (95% CI) for each SNP. Stratified analysis by oestrogen receptor (ER) and progesterone receptor (PR) status was also performed. Among 16 variants, only seven conformed to Hardy-Weinberg proportions in the controls. None of these seven variants showed statistically significant differences between the case and control groups in this Han Chinese population. However, chr6_32737733, a variant in HLA-DQB1, showed significant associations with both ER-negative and PR-negative breast cancer in the best fit to the dominant model. Furthermore, another significant correlation was seen between chr6_32606112, a variant in HLA-DRB5, and PR positivity. These results indicate that although no breast cancer risk variants in HLA class II were found in this Chinese population, HLA-DQB1 chr6_32737733 may be involved in determining a poor prognosis, whereas HLA-DRB5 chr6_32606112 may relate to a good prognosis.

Delay of gap filling during nucleotide excision repair by base excision repair: the concept of competition exemplified by the effect of propolis.

Nucleotide excision repair (NER) consists of a sequence of events including DNA damage recognition, excision of the damage containing oligonucleotide, gap filling, and ligation. We found that gap filling during the repair of ultraviolet (UV)C-induced DNA lesions was inhibited by various compounds, e.g., amoxicillin, and mixtures, e.g., propolis, the materials that could induce oxidative DNA damage in serum-supplemented cell cultures. Such inhibitory effect was also demonstrated by the immunostaining experiment and host cell reactivation assay. In this study, we link the repair of oxidative DNA damage with the inhibition of gap filling. Our experimental evidence includes the following: (1) induction of oxidative DNA damage and inhibition of gap filling were quantitatively correlated; (2) although the repair of UV-induced DNA damage was delayed in the presence of propolis, the repair of propolis-induced oxidative DNA damage proceeded regardless of preexposure to UV radiation; (3) inhibition of gap filling by propolis was absent in base excision repair (BER)-deficient cells; (4) suppression of propolis-induced oxidative DNA damage by ß-carotene abolished the inhibition of gap filling; and (5) inhibition of gap filling was also found with typical BER-inducing agents such as hydrogen peroxide, menadione, and methyl methanesulfonate. We propose that competition may occur between NER and BER, which results in delay of gap filling. Our study reveals the dominancy of BER over NER.