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ABSTRACT: To develop a new fluorescence method for the determination of human sodium glucose cotransporter 2 (SGLT2) activity.
Firstly full length human SGLT2 cDNA was cloned and the recombined plasmid pIRES2-EGFP-SGLT2 was constructed. Then the construct was subjected to restriction enzyme digestion analysis. In addition, SGLT2 insert clones were fully sequenced to confirm its nucleotide sequence, and then the recombined plasmid was transfected into HEK293 cells. The expression of green fluoresscent protein (GFP) was detected by confocal and flow cytometry (FCM), respectively. The protein expression of SGLT2 was determined by Western Blot assay. The transport activity of SGLT2 was determined by FCM choosing 2-NBDG as the detection target.
Both restriction enzyme digestion and DNA sequencing assays showed that the recombined plasmid was constructed successfully. After transient transfection into HEK293 cells, the GFP expression analysis displayed high transfection efficiency and transcription activity, and the fluorescence intensity of the transfected cells was much higher than that of the untransfected cells (P<0.01). SGLT2 was more highly expressed in pIRES2-EGFP-SGLT2 transfected cells as compared with the empty vector (pIRES2-EGFP) transfected cells (P<0.05); the expression of SGLT2 in the untransfected cells was similar to that in the empty vector transfected cells. The Na(+) dependent 2-NBDG uptake was significantly increased in the transfected cells compared with that in the untransfected cells (P<0.01).
A new fluorescence method for determination of SGLT2 activity has been developed with a eukaryotic expression vector of human SGLT2.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 10/2012; 44(5):725-31.
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ABSTRACT: Rhein (RH), a compound purified from Radix et Rhizoma Rhei, has been used to alleviate liver and kidney damage. It is found that RH inhibited the differentiation of 3T3-L1 preadipocytes induced by differentiation medium in a time- and dose-dependent manner. It was revealed that RH downregulated the expression of adipogenesis-specific transcription factors PPARγ and C/EBPα, as well as their upstream regulator, C/EBPβ. Furthermore, the PPARγ target genes that are involved in adipocyte differentiation, such as CD36, aP2, acyl CoA oxidase, uncoupled protein 2, acetyl-CoA carboxylase, and fatty acid synthase, were reduced after to RH. In addition, high-fat diet-induced weight gain and adiposity were reversed by RH in C57BL/6 mice. Consistent with the cells' results, RH downregulated the mRNA levels of PPARγ and C/EBPα, and their downstream target genes in C57BL/6 mice. Taken together, adipocyte differentiation and adipogenesis were inhibited by RH in cultured cells and in rodent models of obesity. The evidence implied that RH was a potential candidate for preventing metabolic disorders.
Journal of Asian natural products research 08/2011; 13(8):714-23. · 0.61 Impact Factor
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Hongwen Zhu,
Dandan Shang,
Miao Sun,
Sunju Choi,
Qing Liu,
Jiajie Hao,
Luis E Figuera,
Feng Zhang,
Kwong Wai Choy,
Yang Ao,
Yang Liu, Xiao-Lin Zhang,
Fengzhen Yue,
Ming-Rong Wang,
Li Jin,
Pragna I Patel,
Tao Jing,
Xue Zhang
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ABSTRACT: X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.
The American Journal of Human Genetics 06/2011; 88(6):819-26. · 10.60 Impact Factor
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ABSTRACT: To investigate the semi-quantitative method for evaluating the lipid accumulation in pancreas, the KKAy mice, a classical type 2 diabetes mellitus model mice, were used and treated with rosiglitazone (Rosi); and the age-matched C57BL/6J mice were used as normal control. Pancreas was fixed quickly for histological examination with HE staining. For the estimation of the lipid accumulation in pancreas, semi-quantitative method was designed: the number and the size of islet, lipid accumulation in islet and in exocrine gland were observed and the integrative score calculated under the microscope, separately. In KKAy mice, the characteristics of the increased amount of islet, the enlarged area of islet, an abundance of large vacuolations, lipid droplets, and fat proliferation were exposed frequently, and the integrative score increased 2.1 folds compared with that in C57BL/6J mice. Meanwhile, the levels of serum glucose, insulin, and triglyceride (TG) were 1.7, 18.0, and 9.0 times as those in C57BL/6J mice, respectively. With the rosiglitazone (10 mg x kg(-1)) treatment, compared with that in KKAy mice, the pancreatic pathological changes were ameliorated significantly, and the integrative score in KKAy + Rosi mice decreased by 28.9%; and the levels of serum glucose, insulin, and triglyceride decreased by 48.3%, 81.3% and 64.1%, respectively. It showed there is a correlation between the pancreatic pathological semi-quantitative score and the values of serum parameters. In conclusion, this semi-quantitative scoring method is simple and objective for the evaluation of lipid accumulation in pancreas of mice.
Yao xue xue bao = Acta pharmaceutica Sinica 06/2011; 46(6):664-8.
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ABSTRACT: PTP1B is a negative regulator of insulin signaling pathway. This study investigated the effects of compound CCF06240, a PTP1B inhibitor, on insulin sensitivity and lipid metabolic abnormalities in vivo and in vitro, respectively. The insulin resistant IRM mouse model was induced by HFD. The responses to insulin were determined by OGTT, ITT, and hyperinsulinemic-euglycemic clamp test. The body weight and the levels of serum TC and TG were measured to estimate the lipid metabolism in vivo. Recombinant human GST-PTP1B protein was used to measure the inhibition of CCF06240 on PTP1B activity. The hepatocyte lipid accumulation was induced by high concentrations of FFA and insulin in HepG(2) cells, and evaluated by the Oil Red O method. In IRM mice, the insulin resistance was improved; the body weight and the levels of TC and TG were also reduced by oral CCF06240 administration. In lipid accumulated model cells, CCF06240 was found to reverse the increased PTP1B activity, enhance the insulin-induced tyrosine phosphorylation in insulin signaling pathway, attenuate the FFA-insulin-induced cellular lipid accumulation, and down-regulate the expressions of genes related fatty acid synthesis. These results demonstrated that the PTP1B inhibitor, compound CCF06240, could increase insulin sensitivity through the regulation of insulin signaling pathway, and decrease FFA-insulin-induced hepatocytes lipid accumulation by reducing fatty acid syntheses.
Molecular and Cellular Biochemistry 05/2011; 357(1-2):65-72. · 2.06 Impact Factor
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M Sun,
F Ma,
X Zeng,
Q Liu,
X-L Zhao,
F-X Wu,
G-P Wu,
Z-F Zhang,
B Gu,
Y-F Zhao,
S-H Tian,
B Lin,
X-Y Kong, X-L Zhang,
W Yang,
W H-Y Lo,
X Zhang
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ABSTRACT: The Sonic hedgehog (SHH) protein produced in the zone of polarising activity (ZPA) is a major determinant of the identity and numbers of digits in early limb development. Preaxial polydactyly types II (PPD2) and III (PPD3) have been mapped to a critical region at 7q36, and subsequently shown to be caused by point mutations in the ZPA regulatory sequence (ZRS), a long range cis-regulator for the SHH gene. Triphalangeal thumb-polysyndactyly syndrome (TPTPS) and syndactyly type IV (SD4) were also mapped to the 7q36 region but pathogenic mutations in ZRS have not yet been affirmed.
We performed linkage and haplotype analysis in six Han Chinese families with TPTPS and/or SD4, and refined the disease locus to an interval of 646 kb containing ZRS. In all families, the affected individuals heterozygous at rs10254391 (a single nucleotide polymorphism within ZRS) revealed a remarkable allele imbalance on sequence chromatogram. Using real-time quantitative polymerase chain reaction (qPCR), we identified duplication of ZRS and found that this duplication segregated with the limb phenotypes in all families but was not detected in unaffected family members or in unrelated control individuals. The duplication was also confirmed by interphase fluorescence in situ hybridisation (FISH) in an affected individual. We designed 17 additional qPCR assays and defined the minimum duplications in all six families, ranging from 131kb to 398kb.
Both TPTPS and SD4 are due to duplications involving ZRS, the limb specific SHH enhancer. Point mutations in the ZRS and duplications encompassing the ZRS cause distinctive limb phenotypes.
Journal of Medical Genetics 05/2008; 45(9):589-95. · 6.36 Impact Factor
