Abstract:Caveolins are a family of plasmalemmal vesicles caveolae-associated integral membrane proteins and a marker protein of caveolae involved in the formation and localization that associated with vesicular transport, cellular cholesterol homeostasis and signal transduction. Recent years, strong experimental evidences indicated that caveolins play a pivotal role in the brain function such as neural development, synaptic plasticity and neurodegenerative diseases. Recent progress on studies of the structure and functions of caveolins was simply summarized. The regulatory role of caveolins in the brain functions has been reviewed and expected.

Abstract:Many kinds of cells responsing to mechanical signals were named mechanocytes, including endothelial cells, fibroblasts, osteoblasts, smooth muscle cells. Stress can cause cellular regulation in gene level, and insulin-like growth factorⅠ (IGF-Ⅰ) is one of the factors sensitive to mechanical stimulation. Through the mechanical stretching to skeletal muscle, it was found that mechanical stimulus led to the generation of two kinds of IGF-Ⅰ isoforms, one of which was named mechano growth factor (MGF). MGF can activate satellite cells, promote the proliferation of myoblasts, and plays an important role in treating the loss of muscle mass, preventing myocardial damage and repairing nerve damage, etc. Mechanical stretch can also cause osteoblasts to express MGF, and studies suggested that cyclic stretching (The strain rate is 15%) applied to osteoblasts increased the expression of IGF-Ⅰ and produced the splicing isoform MGF. The further study on MGF may broad prospects for treating diseases and tissue engineering.

Abstract:Lipopolysaccharide(LPS) can induce cell inflammation through interacting with TLR4. Recent studies have revealed that MD-2 participate in the process of LPS induced signal transduction pathway by forming a complex with TLR4. After binding to the MD-2 of the TLR4/MD-2 complex, LPS can induce TLR4- oligomerization and activate the downstream signal pathway. After being synthesized, most MD-2 can bind to TLR4 at the endoplasmic reticulum /Golgi apparatus and expresse as TLR4/MD-2 complex at the cellular surface. Therefore MD-2 not only can regulate the distribution of TLR4 in the cytoplasm, but also help TLR4 to recognize LPS. Another part of MD-2 can be released into plasma as soluble MD-2(sMD-2). With the help of CD14, sMD-2 would interact with LPS in the plasma to constitute LPS-sMD-2 complex, helping cell who express only TLR4, to recognize LPS, however excessive expressed sMD-2 would repress the LPS signal transduction pathway. In conclusion, MD-2 plays a crucially modulating role in the process of TLR4 mediated endotoxin recognition and signal transduction.

Abstract: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 pchp53_Arg and pchp53_Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53_Arg and CMVe+hTERTp+p53_Pro 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-7402 were 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.

Abstract: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 490 healthy controls was conducted, selecting the ACE gene I/D polymorphism 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.

Abstract: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 methy1 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.

Abstract:The human cellular repressor of E1A-stimulated genes (hCREG), originally cloned from the cDNA library of HeLa cell line, was found to rapidly induce the differentiation of diverse type cells such as pluripotent mouse EC cells, monocytes U937 and myeloid cells EML. It was identified in previous study that not only could overexpression of hCREG regulate and hold the differentiation vascular smooth muscle cells (VSMCs) in vitro, but inhibit the neointima formation in rat carotid artery after balloon injury. These properties suggest that hCREG might have played an important role in antagonizing restenosis of vessel by inhibiting phenotypic modulation of VSMCs. In order to further elucidate the biological functions of hCREG in VSMCs, the upstream molecule mechanisms in regulating its expression were analyzed. At first, bioinformatics was used to predict the hCREG promoter and the binding sites of transcriptional factors. According to bioinformatic results, the pEGFP-hCREG-P945 reporter gene vector was constructed successfully which contained upstream 945 bp of hCREG where two binding sites of E2F1 were determined. Subsequently, the hCREG promoter activity was identified directly by detecting the expression of reporter protein-GFP with fluorescence microscope and Western blot analysis when the vector was transfected into human VSMCs-HITASY. Secondly, both the expression of hCREG and smooth muscle α-actin(SM α-actin) was detected to increase in differentiation HITASY cells cultured for 72 h with serum deprivation，in which the expression of E2F1 was reduced significantly. Inversely, the increase of E2F1 expression was detected in dedifferentiation cells cultured with 10% FBS medium accompanied with the reduction of hCREG and SM α-actin. It is suggested that E2F1 maybe inhibit the expression of hCREG by binding to the hCREG promoter. Furthermore, the E2F1 oligodeoxynucleotide (ODN) and mismatch E2F1 ODN were designed and used to block the binding of E2F1 to hCREG promoter by transcriptional factor “decoy strategy”. Western blot analysis showed that expression of hCREG, GFP and SM α-actin was increased obviously when the E2F1 ODN was transfected in dedifferentiation HITASY cells. The result identified that transcription factor E2F1 inhibited the expression of hCREG and promote dedifferentiation of VSMCs by binding to the sites of hCREG promoter. It can be concluded that E2F1, as a transcriptional regulation factor of hCREG, can repress the expression of hCREG and involve in a pivotal role in the process of VSMCs phenotypic modulation.

Abstract:By means of cDNA amplified fragment length polymorphism(cDNA-AFLP) technique, a fragment P1708 was amplified from Polima cytoplasmic male sterile Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinenesis Makino, syn, B. rapa L. ssp. chinenesis) ‘Bpol97-05A’. RT-PCR showed that this fragment was specifically expressed in male sterile material. Sequencing and BLAST search in GenBank database indicated that P1708 had 100% homolog with chloroplast ndhJ-trnF gene region except a 54 bp insertion. Gene specific primer pairs were synthesized according to ndhJ-trnF gene region and two fragments about 1 900 bp were amplified respectively using genomic DNA templates of Polima cabbage and male fertile oilseed rape. The sequencing results showed that the gene region ndhJ-trnF of Polima cabbage contained two 54 bp repeats and some variation sites. The repeat part shared the same sequence as trnF gene except three bases at 5′ ends. For the insertion of 108 bp sequence, a new open reading frame was created.

Abstract:It has been well known that apoptosis induction and cell cycle arrest are typical biological effects observed in cancer cells after proteasome inhibition. TH2 is a new natural xanthone analogue isolated from the resin of Garcinia hurburyi tree. Here, the cell growth inhibition of TH2 on human hepatocellular carcinoma cell line (Bel-7402) was evaluated in vitro using SRB assay. The treatment of 10 μmol/L TH2 reduced the surviving fraction from 86% (12 h) to 17.2% (48 h). To assess whether TH2 induce apoptosis, the appearance of sub-G1 peak, a specific fraction for apoptosis was detected by flow cytometry analysis. Progressive increase in the percentage of apoptotic population was observed in a dose-and time-dependent manner. Furthermore, a cleavage of poly (ADP-ribose) polymerase (PARP), a marker of early apoptosis, was observed clearly when the cells exposed to 10 μmol/L of TH2 for 24 h by immunoblotting analysis. In vitro activities of 20 S proteasome purified from human erythrocytes on fluorogenic peptide substrates revealed that TH2 inhibited the trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities in dose-dependent manner. Moreover, the turnover of tumor suppressor p53, a sign of deregulation of cell cycle progression and apoptosis induction by classical proteasome inhibitors, was disrupted in Bel-7402 cells. All these data indicate that TH2 had inhibitory effect on the proliferation of Bel-7402 cells and induction of apoptosis, which might be related to its inhibition of proteasome.

Abstract:Proline-rich cell wall proteins are widely spread in plants and are believed to function by modeling the architecture of the cell wall surrounding specific cell types. Five genes encoding proline-rich proteins were isolated from cotton cDNA libraries. The most common characteristic of these proteins is the abundant proline residues that occur in repeating motifs of at least two consecutive Pros. Based on amino acid composition, repetitive motifs and domain organization, the five members can be divided into two subgroups: one group similar to common PRPs including GhPRP3, GhPRP6, GhPRP5 and GhPRP4 was composed of two domains, an N-terminal hydrophobic domain (or signal peptide) followed by a proline-rich domain containing different proline-rich repetitive motifs; the other group different from common PRPs including GhPRPL lies in it contains an N-terminal hydrophilic domain, eight repetitive copies of pentapeptide (similar to PPKKE) lies in the C-terminal domain. Expression studies of the six GhPRPs have been carried out by quantitative realtime RT-PCR. The results showed that GhPRP3 and GhPRP5 were preferentially expressed in 10 dpa fiber, little transcripts was detected in other tissues examined. GhPRPL highly expressed in cotyledons, whereas smaller or negligible amounts of its transcripts were detected in other tissues. The remaining two genes, GhPRP4 and GhPRP6, were expressed in all the tissues analysed, but their transcript level is different. GhPRP4 mRNA is most abundant in hypocotyls, and then in anther, while GhPRP6 expressed highly in fiber, and then in 10 dpa ovule. Furthermore, the results showed that the fiber-specific GhPRP3 and GhPRP5 were also developmentally regulated, suggesting that the genes may play important roles during cotton fiber development.

Abstract:Cationic polymers are being developed quickly as gene delivery vectors. For in vivo gene delivery, the cationic polymers are usually further modified by hydrophilic polymer grafting or ligand conjugation, which have been shown to increase the vector stability, gene delivery efficiency and specificity greatly. Some previous research had shown that modified hydrophilic polymer may partly shield the targeting ligand and result in poor delivery specificity. Developing a method to evaluate the influence of PEG modification on targeting delivery is particularly critical to cationic polymer design and gene therapy development. One of most commonly used cationic polymer polylysine (PLL) was chosen as a model. Targeting ligand epidermal growth factor(EGF)was conjugated with PLL to form PLL-EGF. Then hydrophilic polymer polyethylene glycol (PEG) with molecular mass 7 000 and 20 000 were used to modify PLL-EGF respectively to generate PEG₇₀₀₀-g-PLL-EGF and PEG₂₀₀₀₀-g-PLL-EGF. In BIAcore experiments, epidermal growth factor receptor (EGFR) was conjugated onto BIAcore chip and various PEG modified PLL-EGF solutions were flowed over the chip. By observing the change of RU value, the specific interaction of EGF to EGFR was compared. Compared with PLL-EGF, PEG modified PLL-EGF showed lower association rate and higher disassociation rate to EGFR. Furthermore, compared to PEG₇₀₀₀ modified PLL-EGF, PEG₂₀₀₀₀ modified PLL-EGF got lower association rate and higher disassociation rate to EGFR. The Scatchard analysis results showed that the interactions between EGFR and PLL-EGF or PEG-PLL-EGF are non-linear. It can be concluded that PEG modification indeed reduced the association rate and enhanced the dissociation rate of EGF to EGFR. The length of PEG chain was also a key factor to influence interaction between ligand and receptor. The results showed that it was critical important to evaluate the influence of PEG modification on delivery specificities. The BIAcore method developed in this paper can successfully evaluate the influence, which would be important for cationic polymer design and its application as potential non-viral gene delivery vectors.

Abstract: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.

Abstract:Abnormally hyperphosphorylation of tau plays a critical role in the pathogenesis of Alzheimer disease(AD). Type 2 diabetes whose character is insulin resistance is a known factor of AD. Tau protein were found to be hyperphosphorylated at several AD-related phosphorylation sites (Ser199, Thr212, Ser214, Ser396 and Ser422) in insulin resistant rats. TZD treatment reduced hyperphosphorylation of Ser199, Thr212, Ser396 and Ser422 of tau significantly and of Ser214 of tau to the control level. The activity of GSK-3β was found to be increased dramatically in the hippocampi before and after TZD treatment. These findings suggest (1) that insulin resistance induced by obesity causes a downregulation of insulin signal transduction and the consequent upregulation of GSK-3β activity, which leads to hyperphosphorylation of tau protein, and (2) that rosiglitazone can partially reverse insulin resistance induced tau hyperphosphorylation, which may not mediated by inhibition of GSK-3β activity.

Abstract:To investigate the effect of estrogen to STGC3 gene on CNE2 cell line in vitro and in vivo. The recombinant pcDNA3.1(+)/STGC3 plasmid was constructed and transfected into CNE2 cell line with lipofectamine 2000. CNE2 cell clones with stable STGC3 expression were obtained through G418 selection and identified by RT-PCR and Western blotting methods. The effect of β-estradiol on growth rate of pcDNA3.1(+)/ STGC3/CNE2 cell line was examined by cytometry. The pcDNA3.1(+)/STGC3/CNE2 cell line was inoculated subcutaneously in nude mice. Tumorigenicity diversity was analyzed in female and male nude mice. STGC3 expression was detected in tumor xenografts in nude mice by RT-PCR, immunohistochemistry and Western blotting methods. Cell morphologic changes were observed by microscope. Flow cytometry was used to analyze cell cycles. The results indicated that pcDNA3.1(+)/STGC3/CNE2 cell growth rate decreased after treated with β-estradiol in vitro. There were significant differences in tumor size and mass between pcDNA3.1(+)/ STGC3/CNE2 and control cases (P < 0.05). Furthermore, tumor size and mass had significant differences between male and female nude mice in the STGC3 transfection cases and tumor growth rate of pcDNA3.1(+)/ STGC3/CNE2 in the female nude mice was the lowest in all cases (P < 0.05). No significant difference was found between male and female nude mice in other control cases (P > 0.05). There were more cells blocked in G0/G1 phase in tumor xenografts of pcDNA3.1(+)/STGC3/CNE2 cell line of the female nude mice case than that in other cases. Data above indicated that estrogen might enhance the effect of STGC3 to inhibit CNE2 cell proliferation in vitro and in vivo.

Abstract:At present, transcription analysis of gene expression commonly uses a single housekeeping gene as control for normalization. Through quantitative real-time PCR expression the levels of 6 housekeeping genes(including RPL13A，UBC，EIF4A，B2M，GAPDH，and ACTB) in BEL-7402 cell line were estimated under the role of a new tripeptide compound-YSV. Differences in expression levels were observed by analysis of geNorm program,and RPL13A, UBC were finally determined as suitable internal control genes. In conclusion, the necessity of choosing control genes was proved, and a good way was introduced to select control genes when experiments were handled by different empirical factors(especially under the effect of new materials).