No abstract available.
Animal ; Anions/metabolism ; Blotting, Western ; Chloride Channels/genetics* ; Chloride Channels/analysis* ; Gene Expression/physiology ; Lacrimal Apparatus/cytology ; Lacrimal Apparatus/chemistry* ; RNA, Messenger/analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Submandibular Gland/cytology ; Submandibular Gland/chemistry*

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.
Animals ; Bartter Syndrome/genetics/*pathology ; Chloride Channels/analysis/genetics/*metabolism ; Cloning, Molecular ; Codon, Nonsense ; Dogs ; Humans ; Immunohistochemistry ; Madin Darby Canine Kidney Cells ; Microscopy, Confocal ; Mutagenesis, Site-Directed ; Recombinant Fusion Proteins/analysis/biosynthesis/genetics ; Transfection

BACKGROUNDChronic obstructive pulmonary disease (COPD) is usually complicated with mucus overproduction in airway. Recently the increased expression of the human calcium-activated chloride channel 1 (CaCC(1)) was found to play an important role in mucus overproduction in the asthmatic airways. To investigate the relationship of CaCC(1) and mucus overproduction in the airway of Chinese patients with COPD, the expressions of CaCC(1), MUC5AC and mucus in bronchial tissues were examined.

METHODSBronchial tissues were obtained from fiberoptic bronchoscopy and bronchial biopsy in West China Hospital from April to July in 2004. Twenty-five patients were diagnosed as the patients with COPD overproduction, and other 20 were the control subjects. The expressions of CaCC(1), MUC5AC and mucin in bronchial tissues were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization with digoxigenin (DIG)-labeled RNA probe, immunohistochemical and alcian blue-periodic acid Schiff (AB-PAS) staining, respectively.

RESULTSCompared with the control group, the stronger expressions of CaCC(1) were further detected throughout the bronchial tissues from patients with COPD (P < 0.01). Furthermore, the stronger expressions of the CaCC(1) mRNA were related to the severity of airflow obstruction. Samples from COPD showed a stronger staining for MUC5AC than those in control subjects (P < 0.01) and AB-PAS staining revealed more mucins in COPD patients' submucosal gland comparing with that in control subjects (P < 0.01). Expression levels of the CaCC(1) mRNA were respectively negatively correlated with the patients' forced expiratory volume in one second (FEV(1))/forced vital capacity (FVC) data, FEV(1)% predicted data, V(50)% predicted data, V(25)% predicted data (r = -0.43, r = -0.43, r = -0.35, r = -0.36, P < 0.01, P < 0.01, P < 0.05, P < 0.05). While the expression levels of the CaCC(1) mRNA were well correlated with the expression levels of the MUC5AC mRNA of airway epithelium and the PAS-AB stained area of submucosal glands (r = 0.39, r = 0.46, P < 0.05, P < 0.01). Expression levels of the MUC5AC mRNA were negatively correlated with the patients' FEV(1)/FVC data (P = 0.01), FEV(1)% pred data (P = 0.01), V(50)% predicted data, V(25)% predicted data (r = -0.53, r = -0.53, r = -0.48, r = -0.43, P < 0.01, P < 0.01, P < 0.01, P < 0.01). While the expression levels of the MUC5AC mRNA were well correlated with the positively PAS-AB stained area of submucosal gland (P < 0.05), and the correlation coefficients were 0.43.

CONCLUSIONThese results suggest that the stronger gene expression of CaCC(1) exists, complicated with mucus overproduction in the airway of Chinese patients with COPD.

Adult ; Aged ; Bronchi ; metabolism ; Chloride Channels ; genetics ; Female ; Forced Expiratory Volume ; Gene Expression Regulation ; Humans ; Male ; Middle Aged ; Mucin 5AC ; Mucins ; genetics ; Mucus ; physiology ; Pulmonary Disease, Chronic Obstructive ; metabolism ; physiopathology ; RNA, Messenger ; analysis ; Vital Capacity

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.
Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; chemistry ; genetics ; metabolism ; Brain ; metabolism ; Cells, Cultured ; Chloride Channels ; genetics ; metabolism ; Disease Models, Animal ; HEK293 Cells ; Humans ; Lysosomes ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Microglia ; cytology ; metabolism ; Mutagenesis, Site-Directed ; Peptides ; analysis ; chemistry ; Protein Binding ; RNA Interference ; Sirtuin 1 ; antagonists & inhibitors ; genetics ; metabolism