Human glutamate decarboxylase 65(hGAD65) is an enzyme that catalyzes the transformation of L-glutamic acid into γ-aminobutyric acid.It has been found that Type 1 diabetes mellitus(T1DM)is an autoimmune disease,in which pancreatic islet β-cells are destroyed due to immune response mediated by autoantigen.hGAD65 is considered as a key autoantigen of the autoimmune response, so anti-hGAD65 antibody(hGAD65-Ab) is the most effective and specific immune marker for T1DM diagnosis, and hGAD65 can be used to detect hGAD65-Ab in serum of T1DM patients.The hGAD65 gene was cloned into pET32a(+),then the recombinant plasmid with hGAD65 was transformed into E.coli BL21(DE3) and expressed by IPTG induction.The fusion protein containing thioredox,hexahistidine and hGAD65 (Trx-hGAD65) was mostly insoluble,but the band of soluble Trx-hGAD65 could also be detected by SDS-PAGE,and it was a great improvement compared with the results reported. Trx-hGAD65 was isolated from lysate and purified by immobilized metal ion affinity chromatography(IMAC).After enterokinase digestion and IMAC purification,hGAD65 with high purity was obtained.Detection of thin-layer chromatography(TLC) showed that both Trx-hGAD65 and hGAD65 had enzymatic activity,whereas Trx-hGAD65 had better stability. Furthermore,it was confirmed that Trx-hGAD65 was able to conjugate with hGAD65-Ab in the serum of T1DM patients by ELISA assay.In conclusion,Trx-hGAD65 instead of hGAD65 can be used for T1DM diagnosis,and its application in prophylaxis and therapy of T1DM is expectable.
Objective: To study the role of anti-apoptotic protein Mcl-1 in bile salt (GCDA)-induced chemoresistance of hepatocellular carcinoma cells. Methods: Three HCC cell lines were cultured in CO2 incubator. Expression of Mcl-1 was analyzed by immunofluorescence and Western blot. HepG2 cells were treated by GCDA±CYC or Irinotecan±GCDA. Protein expression and cell viability were assessed by Western blot and MTT, respectively. Drug sensitivity was detected after down-regulation of Mcl-1 by RNA interference in HepG2 cells.Results: Mcl-1 protein is extensively expressed in HCC cells. GCDA prolongs half-life of Mcl-1 from 2-3h to more than 6h and reduces inhibitory action of Irinotecan to Mcl-1. Down-regulation of Mcl-1 by RNA interference can enhance drug sensitivity of HepG2 cells. Conclusion: Bile salt (GCDA) can induce chemoresistance of HepG2 cells. This occurs by increasing protein stability and anti-apoptotic activity of Mcl-1.
Objective: To identify whether serially low density passage could decrease the Oct-4 positive cell percentage and maintain the ability of neurogenesis of mouse ES derived progenitors in expanding stage. Methods: Mouse ES were differentiated into neurons according to fivestep method and given more than 10 continuous passage by low seeding density in proliferation stage. And then cell immunohistochemistry identified Oct-4 positive cells, neurons and astrocytes, flow cytometry tested the Oct-4 positive cell percentage, and the apoptosis testing kit tested the cell apoptosis. Results: The Oct-4 positive cell percentage of mouse ES derived progenitors depressed to 4.33(1.63％ from 16.17(4.8％, cell apoptosis rate of serially low and high density passage in differentiated expanding stage are respective 15.16(3.64% and 11.88(2.63%, and cell immunohistochemistry identified that GFAP and Tuj-1 of the differentiated cells are negative. Conclusion: The Oct-4 positive cell percentage of mouse ES derived progenitors decreased significantly in this differentiation system, and it’s not due to other cells apoptosis was testified. Unexpectedly, the differentiation ability of the progenitors into GFAP positive astrocytes and Tuj-1 positive neurons decreased dramatically at the same time. This result suggested that high density passage play an important part in process of the progenitors differentiated into neurocytes, and the comparision between high and low density culture supplied new platform and the development direction of the neurons differentiation mechanism from ES.
Calsenilin/KChIP3/DREAM, which was originally identified as a presenilin and calcium binding protein expressed at higher levels in nerve system, plays multiple funtional roles as a transcriptional repressor in the nucleus. It binds to the downstream regulatory element (DRE) of prodynorphin or c-fos and its transcriptional repression is modulated by intracellular calcium release. As a voltage-gated potassium (Kv) channels interaction proteins, there are four KchIPs isoform, KChIP1 expressed in variant tissues, KChIP2 specific expressed in heart, KChIP3 and KChIP4 high expressed in brain. They are high homogoly sequence in C- terminus but difference in N- terminus. Because of lacking of the antibody for specific recognize the N-terminus i of calsenilin, it is not clear that the funcrional differents among the KChIP3 family. In other hand, Calsenilin is phosphorylated by CKI（casein kinase I）, PKC, PKA. The multiple phosphorylaton sites containing Ser62, Ser63, and Ser65. Importantly, phosphorylation of the Ser63 oppose its degradation by caspase-3. Calsenilin also called KChIP3 and DREAM appears to process multiple functions in variant tissues by interaction with different moulecules and regulates gene expression, such as depressor the prodynorphin , c-fos , CREB, GnRH , expression in response to calcium influx; regulation of cytokine expression in T-cell proliferation. On the other hand, calsenilin as a activator promote the Vitamin D and retinoic acid response. Thus, calsenilin/ KChIP3/ DREAM as a dual transcriptional regulator on different target molecules. To understaning the functional mechanism of calsenilin/KChIP3/DREAM in aging and oxidative stress response, the functional role in the phosphorylation of calsenilin and relationship between transcriptional repression and phosphorylation of calsenilin in nerve system, the monoclonal antibody gainst human calsenilin was generated by tranditional methods: the human calsenilin was cloned into pGEX-4T-2 vector and expressed by IPTG induction. The purified GST-calsenilin fusion protein was used to immunize mice. PEG-1500 reagent was used to fuse cells. Confocal micrograph results indicate that the monoclonal antibody against calsenilin workes well for Immuno- histochemical analysis, so the overexpressed calsenilin was able to observed in ER/ Golgi complex of H4 neuroglioma cells. Western blot analysis confirmed the monoclonal antibody against human calsenilin reacted on overexpressed protein calsenilin. Ho
