1.Cl- channel expression in choroid plexus epithelial cells.
Peter D BROWN ; Hidetoshi KAJITA ; Aneela MAJID ; Tracey SPEAKE
Journal of Korean Medical Science 2000;15(Suppl):S10-S11
No abstract available.
Animal
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Chloride Channels/metabolism
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Chloride Channels/biosynthesis*
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Choroid Plexus/metabolism*
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Choroid Plexus/cytology
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Epithelial Cells/metabolism*
2.Identification of aging related proteins in human normal colonic epithelium.
Guo ZHU ; Zhi-qiang XIAO ; Zhu-chu CHEN ; Jian-ling LI ; Peng-fei ZHANG ; Yi-xuan YANG ; Xue-ping FENG ; Wei-jian YUAN
Journal of Central South University(Medical Sciences) 2005;30(6):625-630
OBJECTIVE:
To explore the molecular mechanisms of colonic epithelial aging related proteins and aged colonic epithelial susceptibility to tumor.
METHODS:
The proteins of normal human colonic epithelial tissue from young and old people were separated by 2-dimensional gel electrophoresis (2DGE), respectively. Then gels were stained by silver, scanned by imagescanner and analyzed with PDQuest software. The differentially expressed protein spots of colonic epithelium between the old and the young groups were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching.
RESULTS:
Well-resolved and reproducible 2DGE maps of normal human colonic epithelium from the young and the old were acquired. Nineteen more than 2 fold differentially expressed protein spots were identified representing 17 different proteins by MALDI-TOF-MS. The functions of these proteins involve in metabolism, energy generation, transportation, antioxidation, translation and protein folding.
CONCLUSION
Seventeen aging related proteins of human colonic epithelium identified indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium. These data provided useful clues for elucidating the mechanisms of colonic epithelial aging and aged colonic epithelial susceptibility to cancer.
Aging
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metabolism
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Cells, Cultured
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Cellular Senescence
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genetics
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Chloride Channels
;
biosynthesis
;
genetics
;
Colon
;
cytology
;
Electron-Transferring Flavoproteins
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biosynthesis
;
genetics
;
Epithelial Cells
;
cytology
;
Humans
;
Intestinal Mucosa
;
cytology
;
Proteins
;
metabolism
3.Effects of chronic salt loading on blood pressure and arterial chloride channel expression in rats with a two-week-long angiotensin II exposure.
Yuan FANG ; Zhi-Quan LIU ; Yong ZHENG
Journal of Southern Medical University 2008;28(1):7-11
OBJECTIVETo investigate the effects of salt loading on blood pressure and the expression of arterial chloride channel in rats with elevated blood pressure induced by angiotensin II (AngII).
METHODSMale 12-week-old SD rats were randomly divided into AngII and control groups, and in the former group, the rats were exposed to subcutaneous AngII infusion delivered via a drug pump (at 100 ng. kg(-1). min(-1)) for 2 weeks. After the exposure, each group of the rats was further divided into 2 subgroups to receive a high-salt diet (4% NaCl) or normal salt diet (0.6% NaCl) for 12 weeks. The tail blood pressure and sodium metabolism of the rats were measured during the experiment. Since the commencement of salt loading, 6 rats were sacrificed every 4 weeks to obtain the artery samples, in which mCLCA(4) mRNA expression in the arterial smooth muscles was detected by in situ hybridization using mCLCA(4) oligonuclear probe.
RESULTSThe blood pressure of rats in AngII group was significantly higher than that of the control rats (P<0.05), but AngII did not produce significant effects on the expression of mCLCA(4). mCLCA4 mRNA expression was significantly increased in the arterial smooth muscle cells of the rats in high salt groups as compared with those in normal salt groups (P<0.05).
CONCLUSIONA sub-pressor dose of AngII can result in blood pressure elevation, but the mechanism of which does not seem to involve mCLCA(4) expression. mCLCA(4) mRNA expression is up-regulated in normal SD rats after high salt feeding, but salt loading does not obviously affect blood pressure, suggesting the role of mCLCA(4) in antagonizing the pressure-elevating effect of salt loading.
Angiotensin II ; pharmacology ; Animals ; Blood Pressure ; drug effects ; Chloride Channels ; biosynthesis ; genetics ; In Situ Hybridization ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride, Dietary ; administration & dosage ; pharmacology
4.Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function.
Fuxiang ZHU ; Xiandi GONG ; Zelong LIU ; Shude YANG ; Huige QU ; Xiaoyan CHI
Chinese Journal of Biotechnology 2010;26(12):1710-1716
Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
Animals
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Cell Line
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Chloride Channels
;
physiology
;
Cricetinae
;
Cystic Fibrosis Transmembrane Conductance Regulator
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biosynthesis
;
genetics
;
Dependovirus
;
genetics
;
Genetic Vectors
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Humans
;
Membrane Potentials
;
genetics
;
Protein Processing, Post-Translational
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Protein Splicing
5.Expression of three distinct families of calcium-activated chloride channel genes in the mouse dorsal root ganglion.
Mohammed AL-JUMAILY ; Alexei KOZLENKOV ; Ilana MECHALY ; Agnes FICHARD ; Valerie MATHA ; Frederique SCAMPS ; Jean VALMIER ; Patrick CARROLL
Neuroscience Bulletin 2007;23(5):293-299
Objective A calcium-activated chloride current (IClCa) has been observed in medium-sized sensory neurons of the dorsal root ganglion (DRG). Axotomy of the sciatic nerve induces a similar current in the majority of medium and large diameter neurons. Our aim is to identify the molecule(s) underlying this current. Methods Using conventional and quantitative RT-PCR, we examined the expression in DRG of members of three families of genes, which have been shown to have IClCa current inducing properties. Results We showed the detection of transcripts representing several members of these families, i.e. chloride channel calcium-activated (CLCA), Bestrophin and Tweety gene families in adult DRG, in the normal state and 3 d after sciatic nerve section, a model for peripheral nerve injury. Conclusion Our analysis revealed that that mBest1 and Tweety2 appear as the best candidates to play a role in the injury-induced IClCa in DRG neurons.
Animals
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Axotomy
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Chloride Channels
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biosynthesis
;
genetics
;
DNA Primers
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Ganglia, Spinal
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metabolism
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Gene Expression
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Mice
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Neurons, Afferent
;
metabolism
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Reverse Transcriptase Polymerase Chain Reaction
;
Sciatic Nerve
;
physiology
6.Reversal effect of haloperidol on doxorubicin resistance and chloride channel inhibition in erythroleukemic cell K562/Dox.
Chinese Journal of Oncology 2005;27(2):81-85
OBJECTIVETo investigate the reversal effect of haloperidol (Hal) on doxorubicin (Dox) resistance and its inhibition effect on P-glycoprotein and swelling-activated chloride channel in Dox-resistant erythro-leukemic cell line K562/Dox.
METHODSTumor cell proliferation was measured by LDH assay. mRNA expressions of P-glycoprotein (MDR1), glutathione S-transferase Pi (GSTpi) and MDR-associated protein (MRP) of K562/Dox treated with Hal were assayed by RT-PCR. Chloride-sensitive dye MQAE was loaded into K562/Dox cells and the intracellular fluorescence intensity was measured to evaluate the effect of Hal on chloride channel in swelling-activated K562/Dox cells. Coulter counter ZM and Channelyzer 256 were used to measure cell volume regulation.
RESULTSHal significantly reversed Dox resistance in K562/Dox cells after 12.50, 6.25 and 3.12 micromol/L Hal treatment, the chemosensitivity to Dox increased by 8.61, 4.35 and 2.25 times respectively. After treatment with Hal 12.50 micromol/L, MDR1 and MRP mRNA expression were gradually down-regulated in a time-dependent manner on d1-d3, reducing to 76.3% and 64.6% of the control level on d3 (P < 0.05), while GSTpi mRNA expression decreased by 66.1% (P < 0.05) on d1-d2, and began to recover on d3. The swelling-activated chloride channel and cell regulatory volume decreased (RVD) in K562/Dox cells were also inhibited by Hal. Under hypotonic challenge the cellular fluorescence intensity which represented chloride concentration declined by (34.46 +/- 5.91)%. After adding 6.25 micromol/L and 18.75 micromol/L Hal, the hypotonic challenge only caused decrease in fluorescence intensity by (24.43 +/- 3.25)% and (16.63 +/- 4.98)% (P < 0.01). RVD in hypotonic condition was (84.95 +/- 5.69)%. RVD under hypotonic condition with 6.25 micromol/L and 18.75 micromol/L Hal were (51.12 +/- 6.01)% and (39.51 +/- 4.79)% respectively (P < 0.01).
CONCLUSIONA nontoxic concentration of haloperidol can significantly reverse drug resistance through a multi-pathway effect, including down-regulating mRNA expressions of MDR, GSTpi and MRP, inhibition of swelling-activated chloride channel and RVD in K562/Dox cells.
ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Antibiotics, Antineoplastic ; pharmacology ; Chloride Channels ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; genetics ; Haloperidol ; pharmacology ; Humans ; Isoenzymes ; biosynthesis ; genetics ; K562 Cells ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics
7.Effects of Thiazide on the Expression of TRPV5, Calbindin-D28K, and Sodium Transporters in Hypercalciuric Rats.
Hye Ryoun JANG ; Sejoong KIM ; Nam Ju HEO ; Jeong Hwan LEE ; Hyo Sang KIM ; Soren NIELSEN ; Un Sil JEON ; Yun Kyu OH ; Ki Young NA ; Kwon Wook JOO ; Jin Suk HAN
Journal of Korean Medical Science 2009;24(Suppl 1):S161-S169
TRPV5 is believed to play an important role in the regulation of urinary calcium excretion. We assessed the effects of hydrochlorothiazide (HCTZ) on the expression of TRPV5, calbindin-D28K, and several sodium transporters in hypercalciuric rats. Sprague- Dawley rats were divided into 4 groups; control, HCTZ, high salt, and high salt with HCTZ group in experiment 1; control, HCTZ, high calcium (Ca), and high Ca with HCTZ group in experiment 2. To quantitate the expression of TRPV5, calbindin- D28K, and sodium transporters, western blotting was performed. In both experiments, HCTZ significantly decreased urinary calcium excretion. TRPV5 protein abundance decreased in all hypercalciuric rats, and restored by HCTZ in both high salt with HCTZ and high Ca with HCTZ group. Calbindin-D28K protein abundance increased in the high salt and high salt with HCTZ groups, but did not differ among groups in experiment 2. Protein abundance of NHE3 and NKCC2 decreased in all hypercalciuric rats, and were restored by HCTZ in only high Ca-induced hypercalciuric rats. In summary, protein abundance of TRPV5, NHE3, and NKCC2 decreased in all hypercalciuric rats. The hypocalciuric effect of HCTZ is associated with increased protein abundance of TRPV5 in high salt or calcium diet-induced hypercalciuric rats.
Animals
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Biological Transport
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Calcium/urine
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Calcium Channels/chemistry
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Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis
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Hydrochlorothiazide/pharmacology
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Hypercalciuria/*therapy
;
Male
;
Models, Biological
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Rats
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Rats, Sprague-Dawley
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Sodium/*metabolism
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Sodium-Hydrogen Antiporter/chemistry
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Sodium-Potassium-Chloride Symporters/metabolism
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TRPV Cation Channels/*biosynthesis/chemistry
;
Thiazides/*pharmacology
8.Translational Read-Through of a Nonsense Mutation Causing Bartter Syndrome.
Hee Yeon CHO ; Beom Hee LEE ; Hae Il CHEONG
Journal of Korean Medical Science 2013;28(6):821-826
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
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Bartter Syndrome/genetics/*pathology
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Chloride Channels/analysis/genetics/*metabolism
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Cloning, Molecular
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Codon, Nonsense
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Dogs
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Humans
;
Immunohistochemistry
;
Madin Darby Canine Kidney Cells
;
Microscopy, Confocal
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Mutagenesis, Site-Directed
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Recombinant Fusion Proteins/analysis/biosynthesis/genetics
;
Transfection
9.The role of MDR1 gene in volume-activated chloride currents in pigmented ciliary epithelial cells.
Li-Xin CHEN ; Li-Wei WANG ; Tim JACOB
Acta Physiologica Sinica 2002;54(1):1-6
The role of multidrug resistance (MDR1) gene in the activation of volume-activated chloride currents in bovine pigmented ciliary epithelial (PCE) cells was investigated by the patch-clamp technique, the antisense approach, the immunofluorescent technique and the confocal microscopy. PCE cells express P-glycoprotein (P-gp, the product of MDR1 gene). An MDR1 antisense oligonucleotide suppressed MDR1 expression (93% reduction of P-gp immunofluorescence), delayed the activation of a volume-activated chloride current (latency prolonged by 109%), reduced the activation rate by 62% and decreased the peak value of the current by 56%. The transfection reagent lipofectin and the mismatch control oligonucleotide did not significantly affect the current. The data indicate that the volume-activated chloride current is associated with the endogenous expression of MDR1 gene in PCE cells.
ATP-Binding Cassette, Sub-Family B, Member 1
;
biosynthesis
;
Animals
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Cattle
;
Cells, Cultured
;
Chloride Channels
;
physiology
;
Ciliary Body
;
cytology
;
physiology
;
Epithelial Cells
;
metabolism
;
physiology
;
Gene Expression
;
drug effects
;
Genes, MDR
;
physiology
;
Oligonucleotides, Antisense
;
pharmacology