Abstract:The recent investigations have demonstrated that epigenetic such as DNA methylation and histone modification was closely associated with cell growth and malignant tumors, and epigenetic modification was responsible for an important cause of oncogenesis. However, for the recent years some observations have been also shown that the development of tumorigenesis was attributed to transformation expression in microRNA. The latest investigations have revealed that epigenetic was involved in modulation of microRNA expression, on the contrary some kinds of microRNAs could also control epigenetic, moreover, the reciprocal modulation between microRNA and epigenetic could regulate gene expression and induce tumorigenesis. At the same time the data likewise displayed that epigenetic adjusted microRNA expression principally in a way of DNA methylation or histone modification, nevertheless microRNA regulated epigenetic by way of methyltransferases expression, DNA methylation maintenance and histone modification. With regard to the reciprocal modulation between microRNA and epigenetic, a comprehensive and systemic review of reciprocal relationship in modulation of cell growth and oncogenesis was gived.

Abstract:Visfatin is a highly conserved protein expressed by visceral fat tissues that previously identified as pre B cell colony-enchancing factor (PBEF). Visfatin is a 52 ku cytokine and has been shown to exert multiple distinct biological activities. By interacting with insulin receptors, visfatin exhibits insulin mimicking or antagonizing effects under different circumstances. In addition, it possesses an nicotinamide phosphoribosyl transferase (Nampt) activity inside the cells, which functions at the rate-limiting step along the pathway of nicotinamide adenine dinucleotide (NAD) biosyntheses. And finally, as an extracellular cytokine, visfatin is able to induce the expression of inflammatory cytokines, such as TNFα, IL-1β and IL-6. Visfatin has drawn increasing attentions to researchers for its close association with a variety of human metabolic and acute/chronic inflammatory diseases or disorders, including diabetes, obesity, acute lung injury, rheumatoid arthritis, sepsis, myocardial infarction and inflammatory bowel disease. Recently, the SNP (single nucleotide polymorphism) analyses of visfatin and its promoter regions have provided more in-depth understandings of its roles in disease pathogenesis. A discussion in the current knowledge of the structure and diversified functions of visfatin, as well as its connections with a variety of common diseases was given.

Abstract:Apoptosis of neutrophils controls the duration and the intensity of an inflammatory response and therefore the extent of neutrophil- mediated tissue damage, disturbance of neutrophil apoptosis has been associated with many diseases, underlying mechanism is not elucidated. C5a is a complement fragment that has multifunctional properties, which induces neutrophil chemoattraction, an oxidative burst, enhancement of phagocytosis, release of granule enzymes, and suppress neutrophil apoptosis. Several studies have reported calpain is involved in both neutrophil functions and apoptosis and it might play a more specific role in the regulation of neutrophil apoptosis. Diffenrent isoform of calpains is activted by diffenrent stimuli through different transduction pathway. It was reported previously that calpain is required for neutrophil migration and chemotaxis induced by C5a. In addition, autophagy is a ubiquitous physiological process that occurs in all eukaryotic cells and is considered to be a survival mechanism. Atg5 promotes autophagy and is indispensable to autophagosome formation. Upon calpain activation, Atg5 is cleaved and the resulting 24 ku Atg5 mediates apoptosis while losting the property of autophagy. Therefore, Atg5 represents a molecular switch between autophagy and apoptosis. The interaction among the C5a, calpain and Atg5 was introduced and new direction for further research was provided.

Abstract:Using the Tau-1 (or Tau-13) monoclonal antibody, the distribution of endogenetic Tau in the SH-SY5Y cells, HEK-293 cells and HeLa cells have been observed, and the localization of the indirect immunofluorescent signals in both cytoplasm and nuclei in the interphase have been found. In particular, the fluorescent signals were significantly strong in HeLa nuclei. During cell division (metaphase and anaphase), few Tau-1 signals were observed in the nuclei under the same conditions. To confirm the nucleoplasm localization of Tau, the nuclei of SH-SY5Y cells were isolated and it was observed that Tau-1 signals were co-localized with Hoechst33258 (blue) in nucleoplasm using double immunofluorescence. Western-blotting also exhibited tau protein isoforms localized in both cytoplasm and nucleoplasm. It indicates that Tau protein is localized in tumor cells, and also in their nucleoplasm.

Abstract:The 1.2 kb region of 5′ flanking sequence of bovine casein gene and human alpha lactalbumin gene was combined with the PSV vector which has SV40 promotor and β-galactosidase gene(LacZ gene). The expression vector αS1-LA-psv was successfully constructed. Bovine mammary epithelial cell was cultured. The biological characters including survival rate of cells inoculation, doubling time of cell population growth curve and morphology were detected after passaged and purified. And the cell line was identified by the tissue-special expression of cytokeratin 18. The normal cultured dairy bovine mammary epithelial cell line was set up. The cells could still have a good behavior of proliferation after passaged 20 times. The cells were transfected with the eukaryotic expression vector αS1-LA-psv and the activity of β-galactosidase was detected after transfected from 24 h to 120 h. Human alpha lactalbumin was detected at 72 h after transfected too. The production of human alpha lactalbumin was 0.64 g/L approximately. The results indicate that the bovine mammary epithelial cell line have the ability of expressing exogenous gene, the 5′ flanking sequence of bovine αS1 casein gene has the effect of regulating gene expression specifically and the vector αS1-LA-psv which was constructed can coexpress alpha lactalbumin and β-galactosidase.

Abstract:Transient receptor potential A1 (TRPA1) is a cold sensitive cation channel， which could also be activated by various pungent compounds. As a transduction channel in a number of sensory modalities, TRPA1 expressing in heterogonous systems serves to provide great convenience in pharmacological analysis and functional investigation. Due to cellular toxicity, establishment of stable TRPA1 cell line has always been challenging. Nevertheless, the first stable human embryonic kidney (HEK-293) cell line with un-controlled expression of TRPA1 was successfully established. It was also confirmed that this stable cell line retained TRPA1 expression for more than 25 passages in culture. The functional analysis of the cell response verified the stability and specificity of this novel recombinant TRPA1 cell line. Altogether, the data indicated this TRPA1-HEK cell line would be a useful tool for functional analysis of TRPA1 and for the development of high throughput screening (HTS) compatible assay in the effort to identify TRPA1 modulators.

Abstract:SAα2,6 and SAα2,3 linked sialic acid molecules on epithelial cell membrane served as receptors for influenza virus, which are specifically recognized by human and avian influenza viruses, respectively. The distribution of these two species of sialic acids in human respiratory tract from different anatomical sites and different age groups was investigated. The results showed that SAα2,3Gal species was prevalent in respiratory bronchiole and lung alveolar epithelium, but was infrequent in trachea, bronchus and bronchiole. On the contrary, the SAα2,6Gal species was more common in the trachea and bronchus and to a lesser degree in the alveolar epithelium. When compared the expression levels of SAα2,6Gal and α2,3Gal in the respiratory tract among different age groups, no significant difference was found. In the ex vivo H5N1 virus infection study, alveolus epithelium were found to be more susceptible to avian influenza than trachea and bronchus epithelial cells. These results suggest that the human respiratory tract, to some extent, is permissive for avian influenza viruses. The currently-observed limited human to human transmission of H5N1 virus may be associated with the different abundance of SAα2,3Gal linkages in human upper respiratory tract among individuals.

Abstract:Argonaute family proteins are closely linked to small non-coding RNAs (siRNAs, microRNAs, piRNAs). With their functional domains, Argonaute proteins can bind small non-coding RNAs and control protein synthesis, finally affecting mRNA stability, namely RNA interference (RNAi). Moreover, they also participate in the production of Piwi-interacting RNAs (piRNAs). There are 8 members in the human Argonaute family. Using quantitative RT-PCR (qRT-PCR), the expression levels of 4 Ago genes (Ago1～Ago4) were assayed. Experimental results indicated that these genes were ubiquitously expressed in many cell lines. Then it was investigated whether Argonaute subfamily proteins could influence cell cycle regulation by knocking down their expressions in human breast adenocarcinoma MCF-7 cells and cervical carcinoma HeLa cells. The lack of Ago expression resulted in decreased cellular proliferation activity. Further investigation revealed that the cell cycle was delayed at the G0/G1 phases with the especially remarkable arrest occurring in the cases of Ago1 (P < 0.01) and Ago4 (P < 0.05), but without apoptosis. It suggests that Argonaute proteins may function in the cell cycle progression through a possible pathway that does not require small RNAs. The mechanisms underlying this phenomenon are still a puzzle for us.

Abstract:To obtain more information related to the inhibitive effect of anesthetic on spontaneous firing of neurons, several kinds of experimental data were collected before and after applying 10 mmol/L urethane, which included spontaneous firing, sodium current (I_Na), delayed rectifier potassium current (I_K) and transient outward potassium current (I_A) of hippocampal CA1 pyramidal neurons in rats. All of the experiments were performing on hippocampal slices in vitro. Sample entropy (SampEn) and detrended fluctuation analysis (DFA) were employed to measure the complexity and fractal property of spontaneous firing of neurons. It was found that the spontaneous firing rate was significantly decreased, the entropy of ISI sequence was obviously reduced, and the long-range temporal correlations of ISI sequence were weakened after performing 10 mmol/L urethane. Furthermore, I_Na was significantly inhibited, but I_K and I_A were not statistically changed for 10 mmol/L urethane. This suggests that the fractal properties and complexity of underlying dynamics of the system have been reduced by urethane. And the reduction of complexity may because of inhibition of I_Na. The results demonstrate that apparently random fluctuations in neuron spontaneous firing are dictated by a complex deterministic process that imparts “long-term” memory to the dynamic system.

Abstract:Pancreatic acinar cells synthesize pancreatic lipase related protein 1 (PLRP1), which has a high degree of sequence and structural homology with pancreatic triglyceride lipase (PTL). PTL is required for efficient dietary triglyceride digestion, while the physiological role of PLRP1 has not been elucidated, although some investigations have shown that its expression level is changed under some physiological or pathological conditions. Specific antigenic peptides were fused to glutathione S-transferase (GST) and purified recombinant fusion protein was used to generate polyclonal antisera by immunization of rabbits. The antisera could detect the antigen as low as 0.6 ng and PLRP1 protein in 3 μg of mouse pancreatic juice extracts. The high specificity was verified in Western blot and immunohistochemistry analysis by using PLRP1 knockout (KO) mice as the control. Furthermore, it was showed that food intake could increase the exocrine secretion of PLRP1 into pancreatic juice. This implied that PLRP1 may fulfill dietary digestion function in the digestion track.

Abstract:Many somatic cell types were obtained by in vitro differentiation or teratoma formation of human embryonic stem cells (hESCs). However, it is unclear whether specific cell types can be obtained from hESCs-derived teratoma. It was reported that many kinds of cells, including neural progenitor/precursor cells (NPCs) and mesenchymal stem cells (MSCs) were isolated efficiently from the teratoma of hESCs through in vitro selection. The teratoma-derived NPCs and MSCs showed specific characteristics of molecular markers similar to the primary NPCs and MSCs. Moreover, these teratoma-induced NPCs and MSCs can be further induced to differentiate into neurons, astrocytes, or adipose and bone cells, reflecting their inherent multi-potencies. Given that teratoma normally contains a mixture of ectoderm, mesoderm, and endoderm lineage cells at different differentiation stage, it was suggested that hESCs-derived teratoma could be an alternative source to generate a variety of uncommitted progenitor cells or terminally differentiated somatic cells, which may be otherwise difficult to obtain through direct in vitro differentiation.

Abstract:A series of researches concerning the relationship between multidrug transporters and drug resistance in medically intractable epilepsy have been done. There is accumulating evidence demonstrating that P-glycoprotein (PGP) is a candidate to cause AEDs resistance. The effect of PGP inhibitor-verapamil on the intracellular AEDs accumulation in a MDR(multidrug resistant) K562 was investigated. The multidrug resistant (overexpression of PGP) cell line K562/Dox was established and the intracellular PHT and CBZ accumulation in multidrug resistant cell line and non- multidrug resistant cell line was observed. After PGP inhibitor-verapamil was applied to the two cell lines, the concentration change of PHT and CBZ in MDR cell was observed. The results were found: compared with non-MDR cell line K562, which IC₅₀ was significantly increased in MDR cell line K562/Dox after PHT and CBZ was applied; verapamil could decrease significantly the level of IC₅₀ in MDR cell line K562/Dox, and the reversal index were 2.5 and 1.5. The concentration of PHT and CBZ in MDR cell line K562/Dox was lower than that in non-MDR cell line K562, and verapamil significantly increased the concentration of PHT and CBZ in MDR cell line K562/Dox(P < 0.05). It is obvious that overexpression of PGP are concerned with drug resistance of medically intractable epilepsy.

Abstract:To confer the influence of Hsp22 on pathogenesis of SCA3/MJD. GMR-GAL4 and elav-GAL4 system SCA3/MJD transgenic Drosophila models were constructed by using the promoter GMR-GAL4 and elav-GAL4 which drive target selective gene expression in developing eyes and neurons, respectively. Then, Hsp22 protein was overexpressed in SCA3/MJD transgenic Drosophila models at different levels by genetic methods and heat shock reaction. Overexpression of endogen Drosophila Hsp22 can notably suppress the neurotoxicity of MJDtr-Q78 protein, and the level of Hsp22 expression was in consistent with rehabilitation for neurodegeneration of Drosophila eyes, and extension of Drosophila lifespan. It is firstly confirmed that expression of Hsp22 protects the SCA3/MJD from neurodegeneration on Drosophila models, which might contribute to a potential therapeutic effect on SCA3/MJD.

Abstract:Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt cry1Ac gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt cry1Ac gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16 S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin (20 mg/L), 6-BA (1.5 mg/L) and NAA (0.2 mg/L) at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin (20 mg/L) and 6-BA (0.5 mg/L) and NAA (1.0 mg/L) one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide (PPT 10 mg/L) several subcultures. The shoots were transfered into the root-induction medium with herbicide (PPT 10 mg/L) and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity (the mortality of larvaes was 33%～80%) and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.

Abstract:A parallelled purification procedure suitable for acquiring high purified multiple recombinant antigen proteins should be established for construction of antigen protein microarray. An efficient approach for purification of tumor antigen proteins by affinity chromatograph and prepared gel electrophoresis for construction of tumor antigen microarray was established and evaluated. In the procedure, the inclusion bodies were prepared firstly. Then, a Ni-NTA His-bind resins was applied to all dissolved inclusions. The yield and purity of the affinity purified step were 71% and 70% respectively. After the initiative affinity chromatograph purification, the eluted purified recombinant antigen proteins were runned on the prepared scaled SDS-PAGE, and the purified recombinant antigen protein bands were indicated by KCl solution and cutted out. Furthermore, a common dialysis and refolding procedure were applied to the electro-eluted purified recombinant antigen protein solutions. The yield and purity of this further purified step were 32% and 95% respectively. Furthermore, the reaction of purified recombinant RPS4 antigen against the anti-RPS4 autoantibody in esophageal cancer patients' sera was confirmed by ELISA. The 16 purified antigen proteins were pin-printed on the aldehyde glass slides for construction a protein microarray. The reaction of the RPS4 antigen in the microarray against the anti-RPS4 autoantibody in esophageal cancer patients' sera was the same as that assayed in ELISA. The research suggests that affinity chromatograph combining prepared gel electrophoresis was an efficient parallelled purification approach for tumor antigens in construction of microarray.

Abstract:With some conditions, there are 1 384 genes selected from 5 333 possible apoptosis sequences which were mined from IPI and GenBank. After the analysis of their subcellular location, tissue expression remarkably, natural antisense transcripts predict, gene clusters distributed in pathways, protein protein network, some interested things were mined. Some genes are differentially expressed in a tissue type; Some genes are NATS and some gene clusters are in more than one pathway. Meanwhile, one 40-bp oligonucleotide microarray which includes most apoptosis genes was made. With the microarray and samples of HeLaT-NAIF1 and HeLaT-pcDNA3, there were 24 genes differentialy expressed. Perhaps, when the Naif1 was over expressed. PAX2, PDCD8, PACD10, DFFA, CASP7 were also expressed differentially. And there is one mRNA, U58668, neither any gene nor protein information annotation, upregulated during camptothecin-induced apoptosis of U937 cells , also upregulated when the NAIF1 induced HeLaT cell apoptosis(all data is public in http://gpcrome.cbi.pku.edu.cn:2005/chip).