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Altered Redox Status in Erythrocytes From Hypertensive Subjects: Effect of (-)Epicatechin

Progress in Biochemistry and Biophysics.2005;32(3):235-238.

Hypertension is a major problem worldwide. There is much evidence to suggest that reactive oxygen species (ROS) radical may play a role in the development of organ damage associated with cardiovascular disease and hypertension. (-)Epicatechin, a member of tea catechins belonging to flavonoid group, is known to be a potent anti-oxidant.The study has been undertaken to evaluate the effect of (-)epicatechin on markers of oxidative stress: reduced glutathione (GSH) and membrane sulfhydryl (-SH) groups in erythrocytes from hypertensive patients. The effect of (-)epicatechin was also compared with a known anti-oxidant L-ascorbic acid. The erythrocyte intracellular GSH content and membrane -SH group content were significantly (P<0.01) decreased in hypertensive subjects. In vitro incubation with (-)epicatechin caused an increase in GSH and -SH content, the effect was more pronounced in hypertensive erythrocytes. Similar results were obtained with L-ascorbic acid. The observed decrease in the level of GSH and -SH groups in hypertension is an indicator of oxidative stress condition. Observation of an increase in red cell GSH content and the protection of membrane -SH group oxidation by (-)epicatechin in hypertensive subjects is a convincing reason to suggest that high dietary intake of foods rich in catechins may help to reduce oxidative stress and concomitant free radical damage in hypertensive patients.

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Preparation of Microcells For Use in Production of Transchromosomic Animals

Zuyong HE ; Xiuzhu SUN ; Yonghui ZHAO ; Ning LI

Progress in Biochemistry and Biophysics.2007;34(7):770-776.

Microcell mediated chromosome transfer (MMCT) is a challenging technique for introducing exogenous chromosomes into interested mammalian cells. Combined with the somatic cell nuclear transfer technique, MMCT has been employed for producing transchromosomic animals of medical and agricultural value. Producing high quality of microcells is a key step in the success of MMCT. Eamamined by fluorescin staining and Giemsa staining, 0.2 mg/L colcemid was considered suitable for inducing high percentage of micronuclei in A9 (neo12) cells, without causing death of a mass of cells. Microcells were produced by centrifugation of micronucleated A9 (neo12) cells in Percoll density gradient containing 20 mg/L Cytochalasin B at 39 000 g. The resulting mixture of microcells, whole cells, karyoplasts and cytoplast fragments was filtered through 8 μm and 5 μm size membrane pores sequentially to obtain pure preparation of microcells. Microcells were then characterized by Giemsa staining and microcell PCR was first applied for examination of the quality of microcell preparation. The result showed that microcells containing our interest chromosomes-human chromosome 12 were equally distributed in the preparation, the preparation was suitable for use in generation of transchromosomic animals.

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Adeno-associated Virus Serotype 2 Mediated Transduction and Expression of The Human β-Globin Gene in Human Early Fetal Liver Hematopoietic Cells

Xiaojuan SUN ; Jing TIAN ; Mengqun TAN

Progress in Biochemistry and Biophysics.2007;34(5):525-532.

β-Thalassemia is an inheritance anaemia disease due to the defect in β- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent β-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of β- globin gene in vivo were performed and the potential role of AAV2 in β-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human β-globin gene (rAAV2-β-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of β-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-β-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the β-globin gene expression. The results showed that the high titer and purity of rAAV2-β-globin had the ability of mediating β-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the β-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the β-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in β-thalassemia gene therapy.

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Combined Action of ACE Gene I/D and GNB3 Gene C825T Polymorphisms on Essential Hypertension in Northern Han Chinese

Wentao HUANG ; Hongjiang YU ; Xiangfeng LU ; Weiyan ZHAO ; Yuelan WANG ; Dongfeng GU ; Runsheng CHEN

Progress in Biochemistry and Biophysics.2007;34(5):471-478.

Essential hypertension (EH), a complex polygenic disease, is considered to the result of the genetic interaction of multiple gene alterations in concert with environmental factors. Evidences showed that angiotensin-converting enzyme (ACE) gene and G protein beta3 subunit (GNB3) gene are both important susceptibility genes for EH, and that there exists putative biological connection between the two genes in developing hypertension. To investigate whether hypertension was affected by gene-gene interaction between the two genes in the northern Chinese Han population, a case-control association study including 502 hypertensive cases and 490healthy controls was conducted, selecting the ACE gene I/D polymorpinsm and the GNB3 gene C825T polymorphism. Linkage disequilibrium analysis revealed a significant nonrandom distribution only in male hypertensives, indicating that interaction between ACE gene and GNB3 gene may predispose males to the occurrence of hypertension. Multivariate stepwise logistic regression in single locus analysis, with adjustment for common risk factors for hypertension, demonstrated that the OR for DD/ID versus Ⅱ for hypertension among men was significant (OR 1.57; 95% CI, 1.09 ～2.27; P = 0.016) in dominant genetic model. In combination analysis stratified with respect to gender, slightly significant ORs were found after adjustment in males: OR for TT vs CC, 0.11; 95%CI, 0.01 ～0.99; P = 0.049 within ACE DD genotype; OR for DD/ID vs Ⅱ, 1.52; 95% CI, 1.01 ～2.29; P = 0.047 within GNB3 CC+CT genotype. The results suggest that ACE, or a nearby gene, is a male-specific susceptible gene for hypertension, and that there may exist epistatic gene-gene interaction between ACE D allele and GNB3 825C allele.

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p53 Anti-tumor Research in Bel-7402 by Using Human-derived Vector

Zhigang XUE ; Jian LI ; Biao YIN ; Yakun ZHANG ; Xionghao LIU ; Qian PAN ; Zhigao LONG ; Heping DAI ; Kun XIA ; Lingqian WU ; Desheng LIANG ; Jiahui XIA

Progress in Biochemistry and Biophysics.2007;34(5):465-470.

In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53Arg and CMVe+hTERTp+p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.

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Homocysteine-mediated Aberrant DNA Methylation in Vascular Smooth Muscle Cells and Its Potential Pathogenic Mechanism

Yideng JIANG ; Jianzhong ZHANG ; Ying HUANG ; Juan SU ; Jingge ZHANG ; Lizhen WANG ; Xiaoqun HAN ; Shuren WANG

Progress in Biochemistry and Biophysics.2007;34(5):479-489.

Hyperhomocysteinemia, which is an independent risk factor for atherosclerosis, may cause aberrant methylation and dysregulation of gene expression, but the characteristics of the aberrant methylation and its key links involved in its pathogenic mechanisms are still poorly understood. The effect of hyperhomocysteine on DNA methylation in vascular smooth muscle cells, its characteristics and the underlying mechanism of Hcy-induced changing in DNA methylation patterns were investigated. Clinical relevant concentrations of homocysteine was added into the cultured vascular smooth muscle cells of the Homo sapien umbilical vein for 24 h. The level of SAM and SAH was detected by HPLC. The activity of SAH Hydrolase was detected by real-time quantitative reverse transcription-PCR and Western blotting analysis. The level and patterns of DNA methylation were measured by endogenous C-5 DNA methyltransferase(C-5 MT-ase) activity and capacity of genomic DNA to accept methyl groups and methylation-dependent restriction analysis. The results indicated that an increased Hcy concentration induced elevated SAH, declined SAM and the ratio of SAM/SAH, reduced expression of SAH Hydrolase, but increased activity of C-5MT-ase. The methylation status of gDNA analyzed by methyl-accepting capacity of gDNA uncovered a demethylation process in gDNA, or homocysteine-caused hypomethylation in gDNA.With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer C↓CGG sequences to CpG islands. The impacts of different dosage of Hcy showed that the varied detrimental effects of Hcy could be attributed to different concentrations via different mechanisms. In mild and moderate hyperhomocysteinemia, the Hcy may primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism, while in more higher Hcy concentration, the notorious impacts may be more directly caused via oxidative stress, apoptosis, inflammation etc.

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Effect of Phosphorylation on Peptidyl-Prolyl Imide Bond cis/trans Isomerization of Peptides With Xaa-Pro Motif

Zhentai ZHU ; Ming SUN ; Yanting GUO ; Yanmei LI

Progress in Biochemistry and Biophysics.2007;34(6):585-594.

The peptidyl-prolyl imide bond cis/trans isomerization of Xaa-Pro motif in the peptide and protein plays an important role to influence their conformation and function. Here, a series of model peptides including phosphorylated and its unphosphorylated counterparts were designed and synthesized. Preliminary 1H NMR experiments and molecular dynamics (MD) simulation were used to analyze the peptidyl-prolyl cis/trans imide bond isomerization. The data indicated that the side-chain O-phosphorylation of the Xaa residues preceding proline affected evidently the isomerization and thereby regulated the peptides conformations. The charges of the phosphate moiety as well as their steric effects might be the driving force for the conformational changes of these phosphopeptides.Moreover, the obtained most stable multiple configurations and their statistic cis/trans concentration distribution in MD simulation were basically consistent with the NMR experiments, which demonstrated that phosphorylation increased the cis conformation of the peptide and the maximum cis ratio is given while the phosphate group has no negative charge.

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Study on dsRNA Mediated Gene Silencing in Saccharomyces cerevisiae:Suppressing The Expression of GRE3 gene

Yan LI ; Ming YAN ; Kui WANG ; Lin XU ; Wei XU ; Pingkai OUYANG

Progress in Biochemistry and Biophysics.2007;34(3):292-298.

RNA silencing is increasingly employed as an experimental strategy to probe for gene function in several organisms. The purpose of the present study was to test the effect of gene silencing by dsRNA in budding yeast Saccharomyces cerevisiae. GRE3 gene encoding an NADPH-dependent aldose reductase was chosen as an example. A recombinant plasmid psiLENT-GRE3 was constructed based on the pESC-LEU backbone and used to transform S. cerevisiae YPH499. The down regulation of GRE3 gene expression by inducing 1 kb RNA duplex and a 136 bp loop was investigated using reverse transcription- PCR. The results showed that double-stranded RNA mediated gene silencing could be used as a functional tool to decrease the expression level of a specific gene in S. cerevisiae, which would contribute to the understanding of RNA interference in budding yeast.

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High-bone-turnover Osteoporosis and Aortic Calcification in Opg Knockout Mice

Yong XU ; Hua YANG ; Jianou QIAO ; Xihua LI ; Lanzhen YAN ; Long WANG ; Guojiang XU ; Jian FEI ; Jiliang FU ; Zhugang WANG

Progress in Biochemistry and Biophysics.2007;34(3):260-266.

Bone turnover is regulated by local concentrations of cytokines such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL). To explore the in vivo biological function of Opg and the mechanism of osteoporosis due to deficiency of Opg, Opg knockout mice have been generated through homologous recombination. Opg-/- mice exhibit a sharply decrease in bone density and strength as expected. The number of osteoclasts in Opg-/- mice significantly increases. Morphologically, osteoclasts appear more cuboidal in shape in Opg-/- mice than those of wt mice, suggesting that active osteoclastogenesis occurs in the absence of Opg. In consistent with this finding, an increase of osteoblast activity was also observed with accelerated mineral accumulation rate by histomorphometric measurement and elevated serum alkaline phosphatase activity (ALP) in Opg-/- mice. Interestingly, more than 50% of 2-month-old Opg-/- mice manifest medial calcification of aorta with comparable serum concentrations of calcium and phosphorus to wt mice. In conclusion, Opg-/- mice have a high-bone-rurnover type osteoporosis. The aortic calcification in Opg-/- mice is not due to abnormality of calcium and phosphorus metabolism. The mechanism underlying aortic calcification in Opg-/- mice needs to be further investigated.

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Preparation and Characterization of Polyclonal Antibody Against Xenopus PAPC

Ruiying HU ; Peng XU ; Yuelei CHEN ; Xin LOU ; Xiaoyan DING

Progress in Biochemistry and Biophysics.2007;34(2):222-228.

Xenopus Paraxial Protocadherin (PAPC), which was initially identified in a screen for genes present in the Spemann organizer of Xenopus embryos, is required for gastrulation, somitogenesis and otic vesicle formation. In order to investigate its function in various developmental events, an antibody was prepared which could specifically recognize Xenopus PAPC. Glutathione S transferase (GST) expression system was used to express the fusion protein GST-PAPC. Rabbits were immunized with GST-PAPC Western blotting analysis of FL-PAPC transfected HEK 293T cells lysates, which could be specifically blocked by pre-adsorption of prokaryotic expressed GST-PAPC fusion protein. Furthermore, by using immunofluorescence analysis the polyclonal antibody recognized membrane-bound PAPC in FL-PAPC transfected 293T cells and Xenopus animal cap cells. By Western blotting analysis,the endogenous 150 ku PAPC protein was detected in Xenopus embryos using the anti-PAPC antibody. Take together it could be concluded that a polyclonal antibody specifically against Xenopus PAPC was developed.

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