Abstract:Viral ion channels are short transmembrane proteins, which have multifunctional roles in the life cycle of virus.It has been shown to form selective ion channel in host cell membrane.Some ion channel blockers are able to block the channels and inhibit the ion channels activity which may lead to diminished virus production. It suggests that the virus ion channels could be a kind of highly relevant target for antiviral drug action.

Abstract:Id (inhibitors of DNA binding/differentiation) proteins, which inhibit cell differentiation and promote cell proliferation, are negative regulators of basic helix-loop-helix (bHLH) type transcription factor. There are four related members of the Id family called Id1, Id2, Id3 and Id4 in mammalian cells. Id proteins are critically involved in the regulation of cell cycle processes, including development, maturation, growth, differentiation, and apoptosis. Since the identification of Id proteins in 1990, the aspects about the roles of Id played in regulation of gene expression, cell proliferation and differentiation, senescence and tumorigenesis have been widely investigated. Thus, the Id proteins have become important molecules for understanding basic biological processes as well as targets for potential therapeutic intervention in human disease.

Abstract:The process of in vivo patch clamp is to recoding electrophysiological signal of neurons in vivo by whole cell techniques, which is useful approach in physical and pharmacological research. A blind patch-clamp technique in the intact brain is usually adopted. Now a new visual method are reported and termed as two-photon targeted patching, which uses two-photon imaging to guide in vivo whole-cell recordings to individual, genetically labeled cortical neurons, so that subset of neurons are specially focused. Here both two methods were simply reviewed.

Abstract:The individual's embryogenesis, cellular proliferation and differentiation are precisely regulated by various kinds of genes in a time-dependent manner. PcG family is a group of the most important genes that related to system development. HPC2 protein which is encoded by a critical PcG gene, hpc2, associates with other PcG proteins, such as HPH, BMI-1 as well as RING1 and constitutes HPC/HPH PcG complex to maintain the repressed state of homeotic gene, so that it can regulate the body development, cellular proliferation and directional differentiation. Furthermore, HPC2 has also been found to interact with some other proteins, suggesting that HPC2 may possess some other functions. Therefore, further study of hpc2 is not only helpful to the understanding of the mechanism of biological action of PcG proteins and expanding of the knowledge on the regulation of gene expression, but also beneficial to the discovery of potential relationship between PcG family and other signal transduction pathways for better understanding of the cellular signal transduction network.

Abstract:In order to define subcellular localization of SDCT2, expression vector of SDCT2 and EGFP (enhanced green fluorescence protein) fusion protein was constructed and transfected into pig renal tubular epithelial cells LLC-PK1. Laser confocal microscopy showed that SDCT2 protein was almost exclusively located at the basal and lateral membranes of LLC-PK1 cells. When SDCT2-EGFP mRNA was microinjected into Xenopus oocytes, green fluorescence of the fusion protein was only found at the cellular membrane. To further determine subcellular localization signal of SDCT2, its N-terminal and C-terminal sequences were deleted and fused with EGFP. These deletion mutants were transfected into LLC-PK1 and their intracellular distributions were observed. The confocal analysis indicated that the SDCT2 with N-terminal deletion was predominantly distributed in the cytoplasm and also expressed at apical and basolateral membranes. The SDCT2 with C-terminal deletion was almost entirely distributed at the basolateral membrane, nearly not expressed at the apical membrane, and seldom expressed in the cytoplasm. Immunohistochemistry staining revealed that SDCT2 only expressed at the basolateral membrane of proximal renal tubular cells. The above results suggested that the N-terminus of SDCT2 is required for its subcellular localization and the basolateral membrane localization signal of SDCT2 locates in the N-terminal sequence.

Abstract:Mitochondrial DNA is more prone to suffering from extensive oxidative damage than nuclear DNA. Base-excision repair which is well established in mitochondria may be involved in the prevention of nucleotides from oxidative damage. It is necessary to consider which severe damage or inadequate repair mainly contributes to the mutations in mtDNA. Human LO₂ liver cells were exposed to 9 mmol/L alloxan for 1 h and then incubated in fresh culture media for 0, 2, 8 and 24 h respectively. The frequencies of oxidative base damage in mtDNA were measured by a quantitative Southern blot coupled with digestion by the enzymes endonuclease Ⅲ and formamidopyrimiding DNA glycosylase. Next, ligation-mediated PCR (LM-PCR) was performed to map damage to specific nucleotides along a ～100 bp fragment including MTTL1 gene. The addition of alloxan to cultured human cells increased the rate of oxidative base damage and ,by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the lesion frequency decreased to levels slightly higher than normal at 8h and returned to normal levels at 24 h. The result of LM-PCR showed 20 hot spots of MTTL1 gene where nucleotides were receiving a majority of damage. The pattern of oxidative damage is like that of point mutation identified in this gene, suggesting the possibility that oxidative damage mainly contributes to the formation of the point mutation in MTTL1 gene.

Abstract:The gene encoding the cytoplasmic domain of human Fas from 175aa to 304aa was amplified from U937,which was then determined by sequence analysis and cloned into pC4M-FV2E. The resulting Myr-FKBP12-Fas fusion gene was subcloned into pEGFP-N1.Stable cell clones as named 293/ FKBP12-Fas were obtained by transfecting the recombinant plasmid pEGFP-N1/FKBP12-Fas into HEK293,which was following selected by G418. The integration and expression of FKBP12-Fas fusion gene were identified by RT-PCR and Western-blotting respectively. Both the observation of the condensation and margination of the chomatin on nuclear membrane under electron microscopy and the detection of typical “DNA Ladder” proved the apoptosis induced by AP20187. However, FK506 could competitively block such cell apoptosis, indicating that the inducible apoptosis cell model established as above offered an applicable technical platform for the screening of FK506-like small molecular compounds.

Abstract:High ratio infection of Mycoplasma hyorhinis in gastric tumor tissues suggests a possible association between mycoplasma infection and cancer. P37, an outer-membrane bacterial protein from Mycoplasma hyorhinis, increases neoplastic invasivity and metastasis and induces TNF-α secretion from human peripheral blood mononuclear cells (PBMC). To investigate the functional mechanism of P37, yeast two-hybrid system was used to isolate proteins interacted with P37 from a human placenta cDNA library. Among the 2.6×10⁶ transformant clones, one positive clone was obtained. Sequence analysis revealed that the clone is a cDNA fragment from Norpeg and encode a carboxy terminal protein. The interaction between P37 and Norpeg was further confirmed by ELISA and GST-Pull down assay. This result would be useful to study the function of P37 further.

Abstract:The interferons are cytokines with antiviral, antiproliferative and immunomodulatory activities. Among three major distinguished types, interferon-gamma (IFN-γ) is more popular for its specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to express recombinant IFN-γ in E.coli in large amounts, however this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. Here minichaperone (GroEL191～345)-mediated in vitro refolding of recombinant human interferon gamma (rhIFN-γ) in free and immobilized GroEL191～345 systems are studied. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations were determined by using a modification of Lowrys method with bovine serum albumin as reference and total activities of renatured rhIFN-γ were measured by CPE method. The results showed that the presence of GroEL191～345 in the refolding buffer not only enhanced the specific activity, but also promoted the refolding efficiency. With the initial protein concentration of 100 mg/L, the protein yield and total activity of rhIFN-γ assisted by GroEL191～345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhIFN-γ assisted by GroEL191～345 were as follows: refolding temperature 15℃, refolding time 4 h, pH 7.7, initial concentration of IFN-γ 100～200 mg/L and the molar ratio of GroEL 191～345 versus IFN-γ 1∶1～2∶1. Furthermore, the immobilization of GroEL191～345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhIFN-γ were 46.29% and 1.95×10⁷ U/mg respectively even the initial protein concentration was up to 400 mg/L.

Abstract:A T-DNA tagged rice population was generated for functional genomic analysis. Using thermal asymmetric interlaced PCR (TAIL-PCR), 159 sequences flanking the right border of T-DNA were obtained. Among them, 92 sequences contained the right border of T-DNA and flanking rice sequences, and 78 flanking rice sequences were mapped as T-DNA tags on 12 rice chromosomes. Integration patterns of T-DNA tags in rice genome were further analyzed using the 78 T-DNA tags and 169 ones reported previously. At junctions of the right border and flanking rice sequences, 14.6% of T-DNA tags (36) showed 3～74 bp of filler sequences. For the T-DNA tags without filler sequences, 21.3% (45) displayed 3～5 bp of microhomology between the nicked T-DNA right border and flanking rice sequences. Filler sequences and microhomology revealed that both double-stranded break and single-stranded gap repair mechanisms played a critical role in the integration of T-DNA into the rice genome. T-DNA integration preferentially occurred in the A/T-rich regions, mainly in the 5′- and 3′-regulatory regions outside the coding regions and in introns of genes.

Abstract:Electrophoretic migration shift assay shows that human neuronal tau can bind to λDNA, plasmid DNA and DNA fragment of PCR product from human cDNA. One mole of neuronal tau binds to a fragment of 6～10 bp nucleotides. Under atomic force microscope, a necklace complex of tau-DNA has been directly observed. Experiments in immunohistochemistry with a monoclonal antibody Tau-1 exhibit condense nuclear staining of tau as well as diffuse cytoplasmic staining of tau. It suggests that neuronal tau plays an important role in nucleus.

Abstract:Survivin plays key roles in cell proliferation and apoptosis regulation. And it was recognized as a molecular marker for tumor diagnosis and an ideal target for cancer therapy. The significant expression of survivin in activated peripheral lymphocyte was observed. To clarify the signal transduction pathway of the activation of peripheral lymphocyte and resulted in expression of survivin gene, normal human peripheral blood mononuclear cell (PBMC) was cultured with PHA and IL-2 in vitro and the expression of survivin was analyzed by RT-PCR, Western blotting and cell cycle. There were significant transcription of survivin and detectable protein of survivin in PBMC cultured with PHA and IL-2 for 36 h. The expression of survivin increased with the extension of culture time and this expression was cell cycle dependent. AG490 (JAK2 inhibitor) resulted in the cell cycle arrested and inhibited the expression of survivin markedly, and Wortmannin(PI3K inhibitor) and PD98059(MEK inhibitor) also inhibited the expression of survivin to a less extent. The results revealed that the expression of survivin correlated with the activation of peripheral lymphocyte and meant the state of cell proliferation. The JAK-STAT signal transduction pathway mediated by JAK2 was essential for the activation of peripheral lymphocyte and the expression of survivin. And the signal transduction pathway mediated by PI3K and MEK also affected this process to a certain extent.

Abstract:Based on the finding of adipophilin expression with the cellular cholesteryl ester increasing, the aim of study was to investigate the active site of adipophilin in cellular cholesteryl metabolism. Mice peritoneal macrophages were incubated with 80 mg/L OxLDL or 80 mg/L OxLDL plus 1 mmol/L adipophilin antisense oligonucleotides respectively. At various time points, the incubated cell samples were observed with adipophilin immunofluoresence staining, flow cytometric analysis and cellular cholesterol analysis. The results shown, the antisense oligonucleotides treated cells contained significantly lower cholesteryl ester (19.9±1.9) mg/g for 72 h. It was also shown by Oil Red O staining that the experimental group cells had less lipid droplets. During 96 h, expression of adipophilin increased in both groups. At 12 h, expression of adipophilin in OxLDL incubated cells increased quicker than that of the cells in antisense treated cells. At 96 h, the level of adipophilin expression in untreated control was significantly higher than that of antisense treated cells. The amount of OxLDL taking up by the cells was also observed with Ox-r［CL-³H］LDL. During the observation, the amount of Ox-r［CL-³H］LDL taking up increased gradually in both groups, however, from 24 h the taking up amount in antisense treated cells was less than that of untreated control. There was a statistical difference between the two groups from 24 h to 96 h. The activity of ACAT in the two groups was observed as well. From 6 h to 48 h, the relative ACAT activity increased in both groups. The relative ACAT activity kept unchanged from 48 h to 96 h in the two groups. At 48 h, the relative ACAT activity in antisense group was significantly lower than that of control group. The activity of ACAT was related to adipophilin expression but was not a linear relationship. This meant, at least partly, expression of adipophilin was related to cellular taking up of lipoproteins and to ACAT activity. Therefore, it is indicated that adipophilin has a role in metabolism of cellular lipid droplets and adipophilin is associated with ACAT.

Abstract:The cDNA encoding keratinocyte growth factor-2 was isolated from human fetal liver cDNA library and subsequently cloned into the bait protein plasmid pAS2-1.The positive clone was obtained from human fetal liver cDNA library screened by yeast two-hybrid system. After sequence analysis and homology comparison, the candidate protein was identified as human ribosomal protein L22.KGF-2 and the candidate proteins were separately cloned into BD and AD plasmid of mammalian two-hybrid assay and cotransfected into COS-7 cell line. The interaction between KGF-2 and the candidate protein was identified by CAT assay.

Abstract:In order to investigate the varied expression of peroxisome proliferator-activated receptor-γ(PPAR-γ) in hypertrophied ventricle induced by pressure overload, the abundance of PPAR-γ in ventricles of spontaneously hypertensive rats (SHR) were detected. Using age matched Wistar-Kyoto rats (WKY) as control, the body mass (BM), left ventricle (LV) wet mass (WM), interventricular septum (IS) WM and right ventricle (RV) WM from 4-week-old and 16-week-old SHR were detected respectively. Then the protein expression in ventricles of WKY and SHR was detected by immunohistochemistral techniques and Western blot, and the mRNA level was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of LVWM, ISWM and RVWM to BM in 4-week-old SHR were similar to that in 4-week-old WKY (P＞0.05), and the protein and mRNA level of PPAR-γ in ventricles of 4-week-old SHR were also similar to that of age matched WKY (P＞0.05). In 16-week-old SHR, the ratio of LVWM and ISWM to BM were higher compared with age matched WKY (P＜0.01), whereas there was no significant difference in RV (P＞0.05). The protein and mRNA level in LV and IS of 16-week-old SHR were great lower than that of 16-week-old WKY (P＜0.01), while in RV there was no significant difference of protein (P＞0.05), and a little decrease of mRNA (P＜0.05). The findings suggested that the expression level of PPAR-γ in hypertrophied ventricles induced by pressure overload (LV and IS in 16-week-old SHR) was significantly decreased, and the decreased expression of PPAR-γ in ventricles might contribute to ventricular remodeling in hypertension.

Abstract:An efficient and economic method for high quality RNA preparation from plant tissues was established. To avoid RNA degradation, sucrose, potassium chloride and Mg²⁺ were included in the extraction buffer. Plant tissues were lysed in the extraction buffer, then ribonucleases (RNAses) and other proteins were denatured and extracted by phenol/chloroform. After that, the DNA was selectively fractionated from RNA with sodium acetate (NaAc) (pH 5.6). The isolated RNA with this method gave good yield. Results of non-denaturing electrophoresis or formaldehyde agarose gel electrophoresis both showed higher amount of 25 S ribosomal RNA (rRNA) than that of 18 S rRNA. Northern hybridization gave sharp and clear signals. Both plastid gene and nuclear gene were amplified successfully by RT-PCR. These results show that, the RNAs isolated with this method are in good integrity and purity, and can meet the needs of most molecular biological experiments including gene cloning and expression analysis. In this method, phenol/chloroform were used to remove proteins and inactivate RNAses, NaAc (pH 5.6) was used to precipitate RNAs, thus largely reduced the experimental expenses.

Abstract:A Gateway-compatible Agrobacterium sp. binary vector system for high-throughput functional analysis of genes in plants has been produced, but a bottleneck problem using this system for fast and reliable DNA cloning is how to obtain entry clones for the PCR products or other DNA fragments simply, economically and efficiently. To address this problem, the traditional TA cloning and the Gateway recombinant cloning techniques are integrated, and two kinds of TA cloning vectors compatible with the Gateway cloning are constructed. The vectors can be used for cloning PCR products or other DNA fragments and at the same time making entry clones in a simple, economical and efficient way.