Association of mtDNA D-loop polymorphisms with risk of gastric cancer in Chinese population.

The aim of present study was to evaluate the association of common polymorphisms detected in mitochondrial DNA (mtDNA) D-loop region (mononucleotide repetitive D310, single nucleotide polymorphism (SNP) D16521) with susceptibility to gastric cancer (GC) in northwestern Chinese population. A total of 180 GC patients and 218 healthy controls were investigated by using PCR- denaturing high performance liquid chromatography (DHPLC) assay. Genotype and allele distributions and haplotype construction were analyzed in case-control study. We found D310 and D16521 heteroplasmy were significantly different between GC cases and controls (p < 0.05), and D16521 homoplasmy showed association with histological grade of GC (p < 0.05). Haplotype 7C/T, 8C/C and 9C/C had significant association with GC risk implied from analysis of D310 and D16521. Taken together, these findings suggested that mtDNA D-Loop polymorphisms and haplotypes may contribute to genetic susceptibility to GC in Chinese population.

Molecular cloning, tissue distribution and bioinformatics analyses of the rabbit BK channel beta1 subunit gene.

Large-conductance, voltage-dependent and Ca(2+)-sensitive K(+) (BK) channels are composed of pore-forming alpha subunits and the modulatory beta subunits. In smooth muscle, the modulatory beta1 subunits are vital in rendering BK channels function as an important regulator of smooth muscle tone and excitability. In this study, we cloned and characterized the BK beta1 subunit gene from rabbits (New Zealand white) and observed its tissue distribution pattern. The full-length cDNA of the BK beta1 subunit, amplified by 5'-RACE and 3'-RACE, is 1,437 bp in nucleotide containing a 447 bp 5'-UTR, a 385 bp 3'-UTR and a 576 bp open reading frame (ORF) which encodes a peptide of 191 amino acids. Sequence analyses showed that the rabbit BK beta1 subunit cDNA is 90, 84 and 82% homologous with that of human, mouse and rat respectively. The similarity is 86, 83, and 83% at the deduced amino acids level with human, mouse and rat beta1 subunit gene, respectively. Northern blots indicated that the rabbit BK beta1 subunit gene is highly expressed in sphincter of Oddi (SO) and aortal smooth muscle tissues, whereas with relatively lower level of expression in heart and skeletal muscle tissues and with no expression found in tissues of liver, lung, kidney and brain. Bioinformatics analyses indicated that the encoded protein is a membrane protein with two transmembrane helical regions containing four functional domains, one possible PKA phosphorylation site (T14) at the N-terminal and two N-glycosylation sites (N80 and N142) at the extracellular loop. For the first time, we identified and characterized the full-length cDNA sequence of the rabbit BK channel beta1 subunit gene, which will set the basis for further investigation in the transcriptional regulation of this gene.

Sphincter of Oddi dysfunction in hypercholesterolemic rabbits.

BACKGROUND AND OBJECTIVES: The mechanisms that trigger gallbladder evacuation dysfunction, the key risk factor for gallstone formation, have not yet been fully elucidated. The sphincter of Oddi (SO) plays important roles in the regulation of gallbladder evacuation and maintenance of normal hydraulic pressure of the biliary tract. The aim of our study was to investigate the effects of hypercholesterolemia on the motility function of SO and the underlying mechanisms of SO dysfunction (SOD). METHODS: Forty New Zealand white rabbits were divided randomly into the control group fed with standard chow and the experimental (Ch) group fed with a high-cholesterol diet for 8 weeks. Changes in the maximal gallbladder emptying rate, gallbladder evacuation with cholecystokinin-octapeptide (CCK-8) stimulation and SO functions of both groups were measured in vivo; B ultrasound examination was used for dynamic observation of peristaltic movements in vivo; SO pressure was measured using manometry; morphological characteristics were observed by electronic microscope; laser scanning confocal fluorescence microscopy was used to identify changes in [Ca]i and Ca oscillation in primary SO smooth muscle cells (SMCs). RESULTS: Gallbladder cholestasis was observed during early stages of gallstone formation in Ch rabbits. CCK-8 could not improve the gallbladder cholestatic state in Ch group. Passive dilation of SO significantly improved the cholestatic state in Ch rabbits (P<0.05), although the maximal gallbladder emptying rate was still lower than that of the control group. Manometry data indicted a significant increase in the base pressure of the SO low-pressure ampulla segment and high-pressure segment (P<0.05) in Ch group. laser scanning confocal fluorescence microscopy assay data indicated that [Ca]i in SO cells of Ch group significantly increased and were in a state of overload (P<0.05); Ca oscillation signals in SO cells of Ch group were also abnormal. CONCLUSION: Hypercholesterolemia initially induced SOD, leading to increased gallbladder evacuation resistance and cholestasis. We suggested that [Ca]i overload and/or Ca oscillation abnormality potentially play important roles in the pathogenesis of SOD.

Down regulated expression of the beta1 subunit of the big-conductance Ca2+ sensitive K+ channel in sphincter of Oddi cells from rabbits fed with a high cholesterol diet.

Hypercholesterolemia, which is closely related to gallbladder bile stasis, can cause sphincter of Oddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO). Intracellular calcium ion concentration ([Ca(2+)](i)) could influence the tension of SO. The beta1 subunit of the big-conductance Ca(2+) sensitive K(+) channel (BK(Ca)) can enhance the sensitivity of the BK(Ca) channel to [Ca(2+)](i). Absence and decline of the BKCa channel subunit beta1 could lead to many diseases. However, the relationship between hypercholesterolemia and the expression of beta1 subunit is not well understood. In this study, we successfully expressed and purified the rabbit BK(Ca) beta1 subunit protein and prepared its polyclonal antibody. The specificity of the prepared antibody was determined by Western blotting. A SOD rabbit model induced by a high cholesterol diet was established and the expression of the beta1 subunit of SO was determined by immunohistochemical staining and western blotting. Compared with the controls, our results demonstrated that hypercholesterolemia could decrease the expression of the beta1 subunit in the SO cells from rabbits. This indicates that lower expression of BKCa channel beta1 subunit might induce SOD.

[The influence on the BK channel beta1 subunit expression of SO caused by high cholesterol in rabbits].

AIM: To investigate the effects on protein expression of big-conductance Ca(2+)-sensitive K(+) channel (BKca) beta1 subunit caused by high cholesterol in Rabbit Oddi's sphincter (SO) cells. METHODS: The rat-anti-rabbit polyclonal antiserum against beta1 subunits of BKca channel of SO cell was prepared. And the protein expression of BKca channel beta1 subunit of SO tissue was detected by semi-quantitative immunohistochemical staining. RESULTS: The protein expression of BKca channels beta1 subunit of SO tissue in HC group was reduced, and there's statistically significant difference between the HC group and the control group. CONCLUSION: High cholesterol can reduce the protein expression of BK Channel's beta1 subunit in Rabbits' SO which suggests high cholesterol can affect the function of BKca channel.

[Preparation of polyclonal antiserum against beta subunit of rabbit BK channel].

AIM: To prepare polyclonal antiserum against beta subunit of rabbit BK channel in mice. METHODS: Gene encoding the intracellular fragment of rabbit BK channel's beta subunit was amplified by RT-PCR. The GST-beta fusion protein was expressed in E. coli. The fusion protein from PAGE gel was used to immunize BALB/c mice and prepare polyclonal antiserum. The specificity of antiserum was identified by ELISA and Western blot. RESULTS: A unique band about 300 bp was amplified by RT-PCR and was verified to be BK channel beta subunit by DNA sequencing. The SDS-PAGE analysis showed that the M(r) of the fusion protein was about 37,000. The purity of GST-beta fusion protein was over 95%. The polyclonal antiserum against GST-beta fusion protein could recognize both GST-beta fusion protein and beta protein in rabbit tissues. The highest titer of the antiserum was about 1:128,000, as shown by Western blot and ELISA, respectively. CONCLUSION: The gene encoding the intracellular fragment of rabbit BK channel's beta subunit has been cloned. The polyclonal antiserum against beta subunit of rabbit BK channel with high titer and specificity has been prepared successfully.