Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

Mucus overproduction is a clinical feature of asthma. Ca2+-activated Cl- channel 1 (CaCC1) has been identified as a protein that is expressed in intestinal epithelia and that plays an important role in fluid and electrolyte transport. Recently, its mouse counterpart, gob-5, was identified as a key molecule in the induction of murine asthma through mucus overproduction. To elucidate the relationship of CaCC1 to human asthma, we examined CaCC1 expression using real-time quantitative polymerase chain reaction analysis in bronchial tissues from patients with asthma and normal control subjects. The expression of CaCC1 was significantly upregulated in patients with bronchial asthma compared with control subjects. In situ hybridization and immunohistochemical analysis demonstrated that CaCC1 is located in the bronchial epithelium, especially in mucus-producing goblet cells. In vitro transfection of a CaCC1 expression vector into the human mucoepidermoid cell line, NCI-H292, increased mucus production and induced the MUC5AC gene. These results suggest that CaCC1 plays a direct role in mucus production and differentiation in goblet cells and may contribute to the pathogenesis of asthma through its mucus-inducing activity.

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD ؊/؊ myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD ؊/؊ myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD ؊/؊ myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD ؊/؊ myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD ؊/؊ myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were downregulated. Consistent with these gene expression profiles, MyoD ؊/؊ myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD ؊/؊ myoblasts. Therefore, MyoD ؊/؊ myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.apoptosis ͉ cell therapy ͉ microarrays ͉ muscular dystrophy ͉ satellite cell

A near-infrared (NIR) spectroscopic sensing system was constructed on an experimental basis. This system enabled NIR spectra of raw milk to be obtained in an automatic milking system (milking robot system) over a wavelength range of 600 nm to 1050 nm. Calibration models for determining three major milk constituents (fat, protein and lactose), somatic cell count (SCC) and milk urea nitrogen (MUN) of unhomogenized milk were developed, and the precision and accuracy of the models were validated. The coefficient of determination (r2) and standard error of prediction (SEP) of the validation set for fat were 0.95 and 0.25%, respectively. The values of r2 and SEP for lactose were 0.83 and 0.26%, those for protein were 0.72 and 0.15%, those for SCC were 0.68 and 0.28 log SCC/mL, and those for MUN were 0.53 and 1.50 mg/dL, respectively. These results indicate that the NIR spectroscopic system can be used to assess milk quality in real time in an automatic milking system. The system can provide dairy farmers with information on milk quality and physiological condition of an individual cow and, therefore, give them feedback control for optimizing dairy farm management. By using the system, dairy farmers will be able to produce high-quality milk and precision dairy farming will be realized

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Twenty-two pesticides were examined in vitro for their effects on hepatocytes, mitochondria, and microsomes isolated from male rats. Twelve pesticides reduced non-protein sulfhydryl (NPSH) content in hepatocytes to less than 80% of control at a concentration of 10(-3) M. Chlorothalonil and ziram were especially effective, reducing NPSH content at 10(-4) M after 90 min incubation. Among those pesticides, only copper terephthalate and chlorothalonil were reactive with glutathione non-enzymatically and enzymatically, respectively. Lipid peroxidation in hepatocytes was stimulated by four pesticides, namely, chlorothalonil, pretilachlor, ethoprofos, and metribuzin at 10(-3)-10(-4) M. Cell viability was considerably decreased following incubation with chlorothalonil, trichlamide, and ziram. Hepatotoxicity of trichlamide was considered to be associated with its direct adverse effects on mitochondrial energy production, since it uncoupled isolated mitochondrial respiration at 10(-6) M and depleted cellular ATP content prior to cell death. Conversely, chlorothalonil- and ziram-induced hepatotoxicity seemed to be related to their depleting effects on cellular sulfhydryls, since addition of the thiol compound dithiothreitol to the hepatocytes incubation mixture protected cells. With respect to isolated mitochondrial respiration, four pesticides inhibited state 3 and/or state 4 respiration rates at 10(-3)-10(-4) M, whereas seven pesticides uncoupled state 4 respiration at 10(-3)-10(-6) M. With respect to isolated microsomal lipid peroxidation, three pesticides were peroxidative at 10(-3)-10(-4) M, whereas three pesticides were antioxidative at 10(-3)-10(-7) M. Only two pesticides, beta-endosulfan and metalaxyl, had essentially no effects on any parameters tested at 10(-3) M.