AIM: To study the effect of the overexpression of p16 on an anion exchange function of band 3 in HeLa cells. METHODS: The expression of p16 and band 3 in HeLa cells was detected by immunohistochemistry (IHC). The p16 cDNA was subcloned to plasmids pEGFP-C1 by PCR and identified by restriction enzyme digestion and sequencing, and then, the recombinant pEGFP-C1-p16 plasmids were transiently transfected into HeLa cells. The expression of fusion protein in HeLa cells was detected by fluorescence microscope. 6-methoxy-N-(3-sulfopropyl)-quinolinium(SPQ)fluorescent probes were used to detect the anion exchange function of band 3. RESULTS: P16 and band 3 were expressed in HeLa cells. The amplificated p16 cDNA sequence was the same as the report sequence. The transfective efficacy of pEGFP-C1-p16 was above 60%. The anion exchange function increased after the transfection of pEGFP-C1-p16 plasmids. CONCLUSION: p16 facilitates the anion exchange function of band 3 in HeLa cells.

AIM: To study the effect of calcium sensing receptor (CaSR) on icariin (ICA) induced mouse embryonic stem cells ( mESCs) to differentiate into cardiomyocytes in vitro.METHODS:mESCs were cultured to embry-oid bodies ( EBs) by direct suspension method and the differentiation of EBs into cardiomyocytes was induced by ICA.The expression of cardiac specific proteinsα-actinin and cardiac troponin-I ( cTnI) was analyzed by Western blot and immuno-fluorescence.The differentiation rate was determined by flow cytometry.The ultrastructure of the derived cardiomyocytes was further characterized by transmission electron microscopy.The expression of cardiac-specific transcription factors Nkx2.5 and GATA-4,as well as CaSR was detected by Western blot.RESULTS: After induction with ICA, the positive characteristics of myocardial cells appeared in the EBs cultured for 2 d.The expression of cardiac-specific sarcomeric pro-tein actinin (α-actinin) and cTnI showed an overall upward trend by Western blot in different phases of ICA induced differ-entiation.The expression of CaSR, Nkx2.5 and GATA-4 was the highest at an early stage of ICA-induced differentiation. Neomycin (an activator of CaSR) up-regulated CaSR, NKx2.5 and GATA-4 expression in the EBs at early stage of ICA-in-duced differentiation, all of which were reversed by NPS2390 ( an inhibitor of CaSR) .CONCLUSION:CaSR is function-ally expressed in mESC-derived cardiomyocytes, and activation of CaSR is involved in the differentiation of mESCs into car-diomyocytes by facilitating the expression of NKx2.5 and GATA-4.

AIM: Ischemic post-conditioning ( PC) plays an important role in cardioprotection from ischemia /reperfusion ( I/R) injury in the young heart but not in the aging hearts .The physiological and pathological roles of hydrogen sulfide ( H2 S) in the regulation of cardio-vascular functions have been recognized .Whether H2 S is involved in the recovery of PC-induced cardioprotection in the aging hearts is unclear.METHOD:The male Wistar young rats (3-month-old), the aging rats (24-month-old), primary cultured cardiomyocytes and the aging cardiomyocytes induced by D-galactose suffered from I/R (or H/R) and PC.RESULTS:I/R (or H/R) decreased H2S production rate and cystathionine γ-lyase (CSE) expression, aggravated cardiomyocyte damage , apoptosis, myocardial infarct size and oxidative stress, reduced cardiac function, increased the levels of Bcl-2, cleaved caspase-3 and caspase-9 mRNA and proteins, promo-ted the release of cytochrome c and mPTP opening, down-regulated the phosphorylation of ERK 1/2, PI3K, Akt and GSK-3βand mito-chondrial membrane potential , up-regulated the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and inhibited PKC-ε transloca-tion and mitochondrial ATP-sensitive K channels (mitoKATP) in isolated young and aging hearts as well as normal and aging cardiomyo-cytes.PC suppressed myocardial I/R injury in the young heart but not in the aging hearts .Supply of NaHS not only increased PC-in-duced cardioprotection in the young hearts and cardiomyocytes , but also attenuated I/R injury and significantly recovered the cardiopro-tective role of PC in the aging hearts and cardiomyocytes .CONCLUSION:The exogenous H 2 S restores PC-induced cardioprotection through inhibiting oxidative stress via the down-regulation of NF-κB and JAK2-STAT3 pathways and mPTP opening by the up-regulation of ERK1/2-GSK-3β, PI3K-Akt-GSK-3βand PKC-ε-mitoKATP pathways in the aging hearts and cardiomyocytes .These findings provide a novel potential target for the treatment of aging ischemic cardiomyopathy .

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

Objective To evaluate effects of flush fluid of different temperatures on pain, comfort and adverse reactions in patients receiving choledochoscopy via T tube for residual bile duct stones. Methods Patients who underwent choledochoscopy were divided into the experimental group ( 36℃ saline was used as flush fluid) and control group ( room temperature saline was used as flush fluid) randomly. Pain degree, comfort degree, procedure time, the total amount of flush fluid, infusion speed and adverse reactions were recorded and analyzed. Results A total of 100 cases were included, 50 cases in each group. There was no significant difference between the two groups in their education level, working condition, operation methods, T-tube size, necessity for lithotripsy, procedure time, total amount of flush fluid and infusion speed (all P>0. 05). The pain scores were 1. 0(1. 00, 2. 00) and 2. 0(1. 00, 3. 25) (Z=-2. 158,P=0. 031) and the comfort scores were 6. 0(5. 00, 7. 25) and 5. 0(2. 00, 6. 00) (Z=-3. 384,P=0. 001) in the experimental group and the control group, respectively. There was significant difference in the change rate of heart rate during and before procedure between the two groups (-4. 07％± 10. 76％ VS 0. 30％± 10. 23％, P=0. 046) . The incidence of postoperative diarrhea in the experimental group and the control group was 6％( 3/50) and 22％ ( 11/50 ) , respectively (χ2=5. 316, P=0. 021 ) . Conclusion 36℃ saline flush can reduce pain scores, improve comfort degree and reduce the incidence of postoperative diarrhea after procedure in patients undergoing choledochoscopy via T tube.