Abstract:Protein phosphatases are crucial for cell living. Protein phosphatase 2A (PP2A) is an important member of serine/theronine protein phosphatase family, which is involved in almost all biological processes in eukaryotic cell. The 3D structure of PP2A core enzyme and holoenzyme was solved in 2006. These results are significant instructions for us to further understand PP2A structure, interactions between PP2A subunits, and also interactions between PP2A and its binding proteins. In a series of studies, PP2A has been considered as a possible tumor inhibitor, which plays an essential role in tumorigenesis and cell transformation. Subunits composition and crystal structure of PP2A, specific carboxyl-terminal modification of PP2A catalytic subunit, interactions between the three subunits of PP2A, and its biological function as a new tumor inhibitor were summarized.

Abstract:DNA methylation is an important epigenetic system. It plays many crucial roles in the gene regulation. With the development of the high-throughput detection techniques, the bioinformatics study has been an active hot topic in the research of DNA methylation. The major achievements and progress on the prediction of DNA methylation status, the mechanism that the majority of CpG islands are resistant to DNA methylation, the relationship between DNA methylation and other epigenetics, as well as the association between aberrant DNA methylation and the tumorigenesis were reviewed in this article.

Abstract:microRNAs (miRNA), a class of recently discovered small RNA consisted of 22 nucleotides, can regulate gene expression by binding to 3′-UTR of target mRNAs. miRNA can negatively regulate the expression of target mRNA at post-transcription and translation level. It is speculated that nearly 30% of the animal genes are regulated by miRNA. Along with the functional studies of miRNA, it is reported that the two important gene expression regulation factors——transcription factors and splicing factors have direct and/or indirect relationships with miRNA. The relationship between miRNA, transcription factors and splicing factors will provide a new insight of miRNA in understanding miRNA’s function and new clues for therapeutical application.

Abstract:Fibroblast growth factor (FGF)-21 is a new member of FGF family. Recently, it is discovered as a potent glucose regulator and a potential drug candidate for treatment of type 2 diabetes mellitus. However the mechanism of action is not known. Mouse FGF-21(mFGF-21) is the best model for study of the mechanism of action of human counterpart, but function of mFGF-21 has not been reported. The aim of this paper is to study the function of mFGF-21 for glucose regulation. For efficiently production of bioactive FGF-21, a Sumo-His expression vector for efficient expression of soluble recombinant proteins was constructed. A hydroxylamine cleavage site was used to substitute the Sumo protease cleavage site for economic purpose. The mFGF-21 cDNA was cloned from mouse liver and sub-cloned into the Sumo-His expression vector. The mFGF-21 was stably expressed in Rosetta host cells, the expressed products were water-soluble. The Sumo-His-mFGF fusion protein was purified by Ni-NTA Column and subsequently subjected to cleavage with hydroxylamine solution to remove the Sumo-His tag; the mature mFGF-21 was dialyzed against 20 mmol/L Tris buffer (pH 8.0) for re-nature. The mature protein with high purity was obtained. The sequencing result indicated that the mature protein consisted of 182 amino acids. SDS-PAGE gel analysis showed that the protein molecular mass was 24 ku, which was recognized by the polyclonal antibody against FGF-21. The amino acid sequence of mFGF-21 had 80% homology with that of human counterpart which was consistent with the published sequence. To examine the glucose regulation activity of mFGF-21, 3T3-L1 pre-adipocytes were differentiated into adipocytes, glucose up-take activity of mFGF-21 was examined by Glucose Oxidase and Peroxidase (GOD-POD) assay at the 14th day after differentiation when 90% of preadipocytes were differentiated into adipocytes. To validate the glucose uptake assay system commercial available human insulin was used to test the assay system. The result showed that insulin could stimulate glucose uptake of 3T3L1 adipocytes in dose-dependent manner, suggesting the glucose-uptake assay system is valid. mFGF-21 was subsequently tested in this system, the result showed that like human FGF-21 and insulin, mouse FGF-21 could also stimulate glucose uptake of 3T3-L1 adipocytes in dose-dependent manner. To examine time of action of mFGF-21, 1000 nmol/L of mFGF-21 were used to treat differentiated adipocytes for 1, 4, 8 and 12 h respectively, together with the same concentration of insulin and BSA as a positive and negative control. Glucose consumption of the medium was examined. The result showed that both insulin and mFGF-21 had tendency to increase glucose up-take of adipocytes with increment of action time. However, mFGF-21 was more potent and showed stronger time-dependent action, which was in agreement with the function of human FGF-21 as reported previously. BSA did not show any glucose uptake activity as expected. It was conclude that mouse FGF-21 is similar to human FGF-21 and possesses strong bioactivity for glucose homeostasis in 3T3-L1 adipocytes, and therefore can be used as a model for study mechanism of action of human FGF-21. The function of mFGF-21 in glucose metabolism at animal level is remained to be studied.

Abstract:FGF-21 is a new member of FGF family and reported to regulate glucose metabolism, as well as lipid homeostasis, therefore, FGF-21 is a potential therapeutics for treatment of diabetes and metabolic syndrome. However, little is known about its functional receptor(s) and mechanism of action. The aim of the paper is to investigate the functional receptor(s) for FGF-21 in order to understand the mechanism of action of the molecule. Previous result showed that FGF-21 could stimulate glucose uptake in differentiated 3T3L1 adipocytes, but not in pre-adipocytes, suggesting that differentiated 3T3L1 adipocytes express functional receptor(s) for FGF-21. Therefore, the differentiated 3T3L1 adipocyte is a perfect target for searching receptor(s) of FGF-21. 3T3L1 pre-adipocytes were differentiated into adipocytes. The cell membrane of the life adipocytes were incubated with FGF-21 which was labeled with either Flag-tag or biotin, the FGF-21/receptor complexes were cross-linked once they interacted by cross-link reagent BS³. The FGF-21/receptor complexes were immunoprecipitated by either Flag antibody or streptavidin and analyzed by SDS-PAGE. The results showed that FGF-21 could form putative FGF-21/receptor(s) complexes with surface membrane protein of differentiated 3T3L1 adipocytes, not pre-adipocytes. The molecular mass of the complexes is proximate 300～400 ku. The complexes were formed by the FGF-21 labeled with either Flag-tag or biotin, suggesting the complex formation was not influenced by different labeling. To understand the component of the FGF-21/receptor(s) complexes, Western blot assay was used to examine the interaction of the complexes with known antibodies. FGFR-2 was detected within the FGF-21/receptor complexes by specific antibody for FGFR-2. To confirm the specific relationship between FGF-21 and FGFR-2, FGF-21-induced FGFR-2 tyrosine-phosphorylation was studied in 3T3L1 adipocytes. The results showed that although FGFR-2 was expressed in both pre-adipocytes and adipocytes FGF-21 could only enable adipocyte-expressed FGFR-2 tyrosine phosphorylation, which was consistent with glucose-uptake by FGF-21 in this two cell types. FGF-21 not only phosphorylated in situ adipocyte-expressed FGFR-2, but also ectopically expressed FGFR-2 in mouse pre-B cell line BaF3 cells. Sequencing analysis of FGFR-2 cDNA from adipocytes revealed that FGFR-2Ⅲc was the only subtype of FGFR-2 expressed in adipocytes, suggesting that FGFR-2Ⅲc is at least one of the functional receptors for FGF-21 and involved in glucose metabolism in the cells. In order to understand the reason why the same FGFR-2 only functions in adipocyte and not in pre-adipocytes, differential expression of FGFR-2 before and after 3T3L1 cell differentiation was systematically analyzed, the result showed that adipocytes expressed high level precursors of FGFR-2, which were not observed in pre-adipocytes, suggesting FGFR-2 turn-over rate could be a key factor to determine the function the molecule. Alternatively, adipocyte-specific molecules could be another key factor participating the signal transduction of FGF-21.

Abstract:The previous study suggested that spindlin1 is a new tumor related protein which is localized to nuclear and may be involved in regulating cell cycle via TCF-4 pathway. To further study mechanism of spindlin1 function, based on the bioinformatics analysis of spindlin1 structure, A series of spindlin1wild or mutant expression vectors was constructed to determine the key amino acid points for spindin1 localization and function. As previously reported, wild type spindlin1 protein was localized in the nuclei of HeLa cells. Cells transfected with construct either with mutation of Ser14＋Ser84, Ser84＋Ser99 or Ser14＋Ser84＋Ser99 exhibited a cytoplasm spindlin1 expression in a diffused manner, while in those cells transfected with construct with mutation of Ser14，Ser84, Ser99 or Ser14＋Ser 99, the spinlin1 showed a similar subcellular nuclear location as wild type spindlin1. Further TCF-4 reporter assay were performed using these wild type or mutant spindlin1 constructs, and the results indicated that mutation of Ser84 with either Ser14 or Ser99 could abolish the promotion of spindlin1 on TCF-4 reporter activity. All these result suggested that Ser84 of spindlin1, with the cooperation of Ser14 or Ser99 play a key role in it nuclear localization and its function in regulation of TCF-4 pathway.

Abstract:The asymmetric hydrolyzation of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. A catalytic antibody that accelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was successfully elicited against an immunogen consisting of tetrahedral sulfate hapten attached to bovine serum albumin (BSA). The rate constant enhancement factor K_cat/K_uncat was about 1.6×10⁴. The catalytic activity of the catalytic antibody in a reverse micelle reaction system based on sodium bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was studied. Kinetic analysis of the catalytic antibody-catalyzed reaction was found to be possible in this system. Kinetic studies showed that hydrolysis in the microemulsion system follow Michaelis-Menten kinetics. The catalytic antibody can also accelerate catalysis of S-ibuprofen methyl ester in the microemulsion system. Temperature effects, the pH profile, K_m,app and K_cat were determined. The dependence of the catalytic antibody hydrolytic activity on the w_o (molar ratio of water to surfactant) showed a bell-shaped curve, presenting a maximum at about w_o = 21.

Abstract:An anti-CD44 monoclonal antibody, HI313, as a potential tool to treat Acute Myeloid Leukemia (AML) have been developed. The binding affinity of HI313 was measured by flow cytometry; suppression of NB4 cell line proliferation induced by HI313 was measured by MTS; effects of HI313 on cell cycle were assessed by flow cytometry; AML cell differentiation was assessed by detecting cell surface markers CD11b, CD14 and CD15. The results show that anti-CD44 antibody HI313 has relative high affinity to cells both high and low expression of CD44 and effectively suppressed the proliferation of NB4 cell line through blocking of cell cycle progress at G0/G1 phase. Furthermore, HI313 antibody can induce differentiation of cancer cells from AML patients. In conclusion, CD44 is a valid target for AML treatment and HI313 monoclonal antibody is a good candidate for therapeutic antibody development.

Abstract:NAD⁺-dependent glycerol 3-phosphate dehydrogenase (GPD) are rate limiting for glycerol production in Saccharomyces cerevisiae. Recently, the gene CgGPD encoding glycerol 3-phosphate dehydrogenase homologous to GPD genes in other yeasts was cloned from Candida glycerinogenes WL2002-5, an excellent industrial glycerol producer. However, the knowledge about CgGPD expression regulation, especially difference with GPD1 and GPD2 from S. cerevisiae is so less. A functional comparison of CgGPD from C. glycerinogenes with GPD1 and GPD2 from S. cerevisiae was undertaken, using S. cerevisiae gpd1/gpd2 and gpd1 osmosensitive mutants as expression systems. The functions of three indicated genes in S. cerevisiae was characterized for osmoregulation under high osmotic stress and redox regulation under anaerobic condition with various transformants. The results showed that gpd1/gpd2 mutants harbouring CgGPD and GPD1 can restore osmotolerance and increase glycerol production ability under hyperosmotic stress but mutant expressed GPD2 can not. When cells were cultured under the anaerobic condition, the growth pattern of mutants harbouring CgGPD and GPD2 are smilar, however the mutant harbouring GPD1 grows slowly and the growth of the controll supresses thoroughly. Furthermore, gpd1/gpd2 mutant employing either CgGPD or GPD2 can increase glycerol production ability and improve GPD specific enzyme activity in anaerobic incubation, but gpd1/gpd2 mutant expressed GPD1 has no the similar results under the same condition. This indicated that CgGPD may involve in both osmoregulation and redox balance and can complement GPD1 and GPD2 in gpd1/gpd2 mutant.

Abstract:Salmonella enterica serovar Typhi, a gram-negative human enteroinvasive pathogen is the etiological agent of typhoid fever, and is an important experimental model for prokaryote research. DNA microarray technology was widely used in analysis of genomic structures and expression profiles. The genomic DNA microarrays were prepared based on the genomic sequences of S. enterica serovar Typhi. A total number of 4 201 protein encoding genes selected from strains Ty2, LT18 and a z66⁺ wild strain of S. enterica serovar Typhi were amplified by PCR. The products were purified and printed onto the poly-L-lysine coated chip slides in duplicate to form the genomic DNA microarrays. Fluorescently labeled probes were prepared by priming of genomic DNAs with random hexamers and extension with Klenow DNA polymerase. Labeled DNAs were hybridized with the microarrays to check the printing effect and verify the genes order. The genomic DNA microarray-based bacterial gene expression profiling analysis was optimized and used to investigate global transcriptional responses when wild strain of S. enterica serovar Typhi was grown in high- and low-osmotic environmental conditions. The major results were consistent with those of previous research with oligo-genomic microarrays of S. enterica serovar Typhi. These results demonstrated that S. enterica serovar Typhi genomic DNA microarrays were successfully prepared and could be utilized in relative gene expression profiling analysis and comparative genomic researches.

Abstract:Two human esophageal cancer cell lines (KYSE-510 and OE33) were chosen as the tumor model to explore molecular mechanism of flavones and flavonols on induction of apoptosis. Effects of flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) on induction of apoptosis in KYSE-510 cells and OE33 cells were observed by DNA fragmentation, acridine orange staining and flow cytometry analysis. The results of real-time RT-PCR and Western-blot analysis showed that the treatment of KYSE-510 cells and OE33 cells with flavones and flavonols induced the expression of PIG3 at the mRNA and the protein levels. Western-blot analytical results further showed that induction of PIG3 caused apoptosis in both esophageal cancer cells through the mitochondrial pathway in a p53-independent manner, and p63 and p73 may be responsible for the induction of PIG3.

Abstract:Human cytomegalovirus glycoprotein complex Ⅱ(gCⅡ) consists of two glycoproteins, gM and gN. Although gCⅡ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32～38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.

Abstract:DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-AZA-CdR on the development of preimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR (0.2, 1.0, or 5.0 μmol/L). Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0 μmol/L) or 4-cell embryos (5.0 μmol/L), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0 μmol/L) or 3/4-cell (5.0 μmol/L) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0 or 5.0 μmol/L) than that in control embryos ( P < 0.05). These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An annexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae ( P < 0.05) in a dose- dependent manner, whereas no significant change was detected in 2- or 4-cell embryos ( P > 0.05). The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to be mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.

Abstract:Polyamine biosynthesis is controlled primarily by ornithine Decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). To study the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis and esophageal cancer cell apoptosis, adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using MTT. The malignant phenotype of Eca109 cells was assessed by growth curve. Western blot and HPLC were used to detect ODC and AdoMetDC expression and polyamine content in Eca109 cells. TUNEL was used to analyze cell apoptosis. The change of morphology of apoptotic cells was observed by electron microscope. It was demonstrated approximate 70% of Eca109 cells were infected with Ad-ODC-AdoMetDCas when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. Ad-ODC-AdoMetDCas could inhibit Eca109 cell growth and invasive ability. TUNEL proved that Ad-ODC-AdoMetDCas can lead to cell apoptosis. Characterized morphology was observed by electron microscope(chromatin condensation, nuclear disintegration, formation of apoptotic bodies). It was suggested Ad-ODC-AdoMetDCas has significant inhibitory effects on esophageal cancer cell proliferation , leads to cell apoptosis and bears therapeutic potential for the treatment of esophageal cancer.

Abstract:Differential gene expression under particular conditions is tightly controlled at the transcriptional and post-transcriptional level. Transcription factors (TFs), key regulators at the transcriptional level, constitute a large and diverse group of double-strand DNA binding proteins which bind close to a target gene to activate or repress its transcription. Large-scale profiling of active transcription factors is essential to assist investigation into complicated gene regulatory networks as well as to help global pathway mapping. Here, a reproducible and reliable high-throughput mouse oligonucleotide array-based platform which can simultaneously explore the activities of nearly 200 different transcription factors is presented. The array comprises 240 synthetic probes which contain TF binding sequences based on 226 PSSMs provided by the TRANSFAC® database. The binding specificity between TF and its binding sequence was validated using antibody-based individual TF assay for AP1, HNF1, HNF4, NF1, NFκB, PPARγ and STAT1. Then purified NFκB was used to test the sensitivity and quantitative ability of the microarray. It could detect as little as 0.5 nmol/L of NFκB protein and maintain linear increase from 0.5 nmol/L to 20 nmol/L. Reproducibility was also explored using nuclear extracts from mouse macrophage RAW264.7 cells. The correlative coefficient between two independent experiments was 0.99 when using the identical nuclear protein. The reliability of the platform was then validated using two well studied cell models including LPS-stimulated MAPK pathway and IFNγ-stimulated JAK/STAT pathway. And finally, the TF profiling microarray was applied into the study of mouse preadipocyte 3T3-L1 differentiation. In addition to the well studied C/EBP family and PPAR family, two unknown transcription factors, SRF and NFAT, were also found changed in the process of adipocyte differentiation. Totally, a reliable microarray platform to profile the mouse transcription factor activities was successfully constructed.

Abstract:The technology of lectin microarrays was established for glycoprotein analysis and initially applied to analyze the glycopattern of whole cell extraction of Chang's liver cells. ConA and GNA were immobilized on the epoxysilane-caoted slides, the standard glycoprotein RNaseB was labeled with Cy3 fluorescent dye, the detection system of lectin microarrays was established for glycoprotein detection and analysis based on the principle of lectin to glycan binding affinity. The consequence of experiment indicated that phosphate buffer containing 1% BSA was the optimal blocking buffer, the optimal incubation time and temperature as well as incubation buffer were 3 hours, room temperature and phosphate buffer containing 1% BSA and 0.05% Tween-20, respectively. Additionally, the specificity of lectin microarrays was validated through the mannose competition assay. Further, the lectin micoarrays containing 10 lectins were fabricated and used to detect and analyze the glycan construction of RNaseB and Fetuin, the result verified the feasibility of our homemade lectin microarray. Eventually they were initially applied to analyze the glycopattern of whole cell extraction of Chang's liver cells. The results indicated that some glycan structure such as multivalent Sia or GlcNAc, terminalα-1,3 mannose, GalNAc and Galβ-1,4GlcNAc possibly existed in the whole cell extraction of Chang's liver cells. Glycosylation is one of the most significant posttranslational modifications of proteins, which plays an indispensable role in a wide variety of biological processes including bacterial infection, cell differentiation, tumor metastasis and cell concretization. Hence, the study on glycosylation draws the attention of researchers widely, but the process develops slowly due to lacking a method can investigate protein-carbohydrate interactions in a rapid, exact and high-throughput manner. The coming lectin microarrays possess the requests above and will promote the process of glycosylation research.