Airway occlusion by mucus in chronic obstructive disease (COPD) is associated with a poor prognosis. We hypothesised that tiotropium has the ability to inhibit neutrophil elastase (NE)-induced goblet cell metaplasia in mice and mucin production in vitro.On days 1, 4, and 7, tiotropium or vehicle was administered to C57BL/6 mice by inhalation and they were allowed to intranasally aspirate human NE. Bronchoalveolar lavage fluid (BALF) and lung sections were analysed on days 8, 11 and 14. The effect of late administration of tiotropium on the goblet cell metaplasia by NE aspiration was also assessed. NE-induced MUC5AC production by NCI-H292 cells was measured with ELISA.Repeated NE aspiration induced marked goblet cell metaplasia. The grading of goblet cell metaplasia, neutrophil count and eosinophil count in BALF, keratinocyte-derived chemokine level and leukotriene B 4 level in BALF, and M 3 receptor expression by immunohistochemistry, were lower in the tiotropium group than in the vehicle group. Late administration of tiotropium inhibited the established goblet cell metaplasia. Tiotropium inhibited NE-induced MUC5AC production.Tiotropium inhibited NE-induced goblet cell metaplasia and mucin production, probably mediated by suppression of inflammation and a direct action on epithelial cells. This result suggests that tiotropium may be useful for the treatment of mucus overproduction in COPD.

Zinc finger fusion proteins, having a Ca-binding site from troponin C, were created to develop Ca-responsive regulation of DNA binding. The typical zinc finger folding of a novel fusion protein with a single finger, F2-Tn, was investigated using UV-vis spectroscopy of the Co-substituted form and CD experiments. Detailed structural analyses of F2-Tn/Zn2+ using NMR experiments and structural calculations clarify that our fusion protein gives a native zinc finger folding with the artificial Ca-binding domain intervening two helices. The Ca-responsive DNA-binding affinity of troponin-fused protein with two fingers (using F1F2-Tn) was investigated by electrophoretic mobility shift assay (EMSA). EMSA analyses of F1F2-Tn were performed under the conditions of various concentrations of the Ca ion. F1F2-Tn has a Kd value of 5.8 nM in the absence of Ca ion and shows a higher Kd value of 13 nM in the presence of 100 equiv of Ca ion. The artificially designed fusion zinc finger protein with a Ca-binding domain has Ca-responsive DNA-binding affinity. It is leading to a better understanding of the construction of zinc finger-based artificial transcriptional factors with a Ca switch.

Summary a) In the course of our polygraphic study of sleep, a new method of EEG analysis was developed. As the result of a simple modification of the ordinary Walter type frequency analyser. we succeeded in obtaining a compact and easily observable record showing the long time variation of the integrated values of each band respectively. b) The application of this method to the study of sleep EEG opened a new aspect. 1. The most remarkable feature of this analysis is the undulatory fluctuation of relatively long time in the low frequency component (long undulatory fluctuation), especially in 1–2 cps band. In general, one sleep cycle is composed of a few of these undulatory fluctuations repeatedly appearing and the state of slight fluctuation mainly corresponding to the paradoxical phase. This long undulatory fluctuation corresponds to successive sleep phases in certain rule. The gradual change appearing at the time of a shift from the moderate phase to the deep phase is characteristic. 2. Generally the crest of the highest of these undulatory fluctuations during successive sleep cycles is highest at the first cycle and tends to decrease toward morning. The increasing slope is steepest at the first cycle and gentler toward morning. 3. During the ascending stage of this undulatory fluctuation the gross body movements are very few and in the other periods are relatively many. Most of the abrupt decreases of the integrated values of the low frequency band are accompanid by gross body movement. This report is dedicated to Professor Haruo Akimoto, Director of the Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, in commemoration of his sixtieth birthday. This study was carried out with the earnest cooperation of C. Sasaki, B.A. and N. Nagamura, M.A. We are much indebted to Mr. K. Akagi of San'ei Instrument Ltd. for the technical support.

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