Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Induction chemoradiation in stage IIIA/N2 non-small-cell lung cancer: a phase 3 randomised trial

BACKGROUND One of the standard options in the treatment of stage IIIA/N2 non-small-cell lung cancer is neoadjuvant chemotherapy and surgery. We did a randomised trial to investigate whether the addition of neoadjuvant radiotherapy improves outcomes. METHODS We enrolled patients in 23 centres in Switzerland, Germany and Serbia. Eligible patients had pathologically proven, stage IIIA/N2 non-small-cell lung cancer and were randomly assigned to treatment groups in a 1:1 ratio. Those in the chemoradiotherapy group received three cycles of neoadjuvant chemotherapy (100 mg/m(2) cisplatin and 85 mg/m(2) docetaxel) followed by radiotherapy with 44 Gy in 22 fractions over 3 weeks, and those in the control group received neoadjuvant chemotherapy alone. All patients were scheduled to undergo surgery. Randomisation was stratified by centre, mediastinal bulk (less than 5 cm vs 5 cm or more), and weight loss (5% or more vs less than 5% in the previous 6 months). The primary endpoint was event-free survival. Analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00030771. FINDINGS From 2001 to 2012, 232 patients were enrolled, of whom 117 were allocated to the chemoradiotherapy group and 115 to the chemotherapy group. Median event-free survival was similar in the two groups at 12·8 months (95% CI 9·7-22·9) in the chemoradiotherapy group and 11·6 months (8·4-15·2) in the chemotherapy group (p=0·67). Median overall survival was 37·1 months (95% CI 22·6-50·0) with radiotherapy, compared with 26·2 months (19·9-52·1) in the control group. Chemotherapy-related toxic effects were reported in most patients, but 91% of patients completed three cycles of chemotherapy. Radiotherapy-induced grade 3 dysphagia was seen in seven (7%) patients. Three patients died in the control group within 30 days after surgery. INTERPRETATION Radiotherapy did not add any benefit to induction chemotherapy followed by surgery. We suggest that one definitive local treatment modality combined with neoadjuvant chemotherapy is adequate to treat resectable stage IIIA/N2 non-small-cell lung cancer. FUNDING Swiss State Secretariat for Education, Research and Innovation (SERI), Swiss Cancer League, and Sanofi.