Abstract:The prevailing sequences in the massive genome sequences, which do not code for proteins or RNAs, have been named junk DNA. It is unreasonable for organisms，in which there is the principle of maximum economy，to accumulate nonfunctional garbage in vital living cells during their long-term evolution. Recent researches have demonstrated that so-called junk DNA possesses important functions and it is believed that more and more junk DNA will be proved to be not garbage but the treasures of the genome.

Abstract:Recent studies have shown that enveloped virus might adopt a similar molecular mechanism of fusion in which two types have been proposed. In TypeⅡ，flavivirus is examples, its fusion mechanism is not similar with typeⅠ and is not understood enough. In TypeⅠ which is the subject of this review and HIV and influenza are its good examples, the attachment glycoprotein of virus binds receptor/s and triggers the conformational change of the fusion protein (attachment protein and fusion protein could be one with two subunits), finally, adopts its most stable fold, the trimer-of-hairpins. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infection cycle.The fusion mechanism involves an intermediate conformational state that can be targeted by therapeutic strategies. Holding the fusion process in the middle would stop the virus entry. The potent and effective therapeutic interventions of virus entry should be possible from a recent clinical trial success of a peptide inhibitor for HIV, Enfuvirtide or T20.

Abstract:RNA interference (RNAi) is the process that the introduction of exogenous or endogenous double-stranded RNA (dsRNA) into a cell leads to special degradation of homogenous mRNA and suppression of relative gene expression. Because of being able to highly specially and efficiently silence gene expression, RNAi has the potent application in the study of gene function and regulation, signaling pathway, drug target validation and gene drug development. RNAi possible molecular mechanism, molecular biological characteristics, productive ways and its application in studying tumor are summarized.

Abstract:Melanocortin 1-receptor (MC1R) gene is a key gene in melanogenesis in mammal. Three single nucleotide polymorphisms (SNPs) were detected in MC1R gene exon using PCR-SSCP and DNA sequencing in chicken. Then these SNPs were analyzed in a CAU three-generation chicken population whose founder population are broiler and white-silk chickens. The result of these analysis indicated that the polymorphisms of the amplified fragment of primer pair 3 is due to a single point mutation G867A, and primer pair 5 is due to two other single point mutations C1292T and C1377G. Lastly, the relations between SNPs and chicken melanin traits were analyzed, the results showed that there was significant correlation （P＜0.05）between the G867A mutation and chicken skin color , between the C1292T mutation and chicken live body shank color and between the C1377G mutation and chicken meat color. The results of the study indicated that MC1R gene is the candidate gene that regulates melanin traits in chicken.

Abstract:In order to determine the importance of hhlim in cardiomyocyte differentiation and to define the hierarchy of transcription factors during cardiac development, the cardiac differentiation profile of the pluripotent P19 cells was characterized at cellular and molecular level. Genes that express early in the cardiogenesis of the embryo include GATA-4 and Nkx2.5. The time course of expression of these genes during in vitro differentiation of P19 cells into cardiomyocytes was established using RT-PCR. GATA-4 is expressed at day 2 after treatment with DMSO, and Nkx2.5 at day 3. hhlim is almost undetected until day 4 after DMSO treatment. Western blot had the same results, which showed that hhlim might participate in the cardiac differentiation pathway. P19-hhlim-overexpressing cells did not show morphological or biochemical evidence of cardiomyogenesis without DMSO treatment, but overexpression of hhlim had a benefit effect on cell aggregates that were especially obvious in the absence of DMSO. Moreover, when hhlim-overexpressing cells were treated with DMSO, the expression of Nkx2.5 and GATA-4 appeared earlier compared with the control. hhlim-deficient P19 cells induced by DMSO retained the ability to differentiate into cardiomyocytes. The time course of expression of Nkx2.5 and GATA-4 in the hhlim-deficient clones was significantly reduced, whereas these clones treated by RA produced neuroectodermal derivatives. Then it was concluded that lack of hhlim expression specifically inhibits cardiomyocyte formation of P19 cells without affecting neuronal differentiation pathway. These data showed that overexpression of hhlim could enhance cardiogenesis of embryonic stem cells whose mechanism might be involved in upregulation of the expression of Nkx2.5 and GATA-4.

Abstract:Cancer and embryo expression protein 65(CEP65) specifically reacted with Mab3H11, which can bind specifically to different cancer cells from different tissues. Northern blot and RT-PCR showed that the mRNA of CEP65 was extensively distributed in embryonic tissue and different cancerous tissues, but not in corresponding normal tissues. In order to study the function of CEP65 in tumor development, using the yeast two-hybrid system, with CEP65 as a bait, a placenta cDNA expression library was screened, and fourteen positive clones was isolated in yeast from 5.2×10⁶ clones. To ascertain the interaction, the 51st clone which interacted more intensive with CEP65 in yea was selected to do GST pull-down assay with CEP65. The result indicated that CEP65 also interacted with the 51st clone in vitro. Bioinformatics analysis showed the interaction between CEP65 and the 51st clone maybe regulate the cell differentiation and proliferation by WW domain or protein kinase C.

Abstract:The mature differentiation of ES cells into hepatocytes and its differentiation ratio were studied in vitro. First, the BALB/c mouse ES cells were cultured for 5 days to develop into EBs in EBs culture medium. Then, the cell growth factors such as transform growth factor (TGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were added into the medium to induce the differentiation of EBs cells into hepatocytes. During the culture medium, the reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and radioimmunoassay (RIA) were used to determine the expression of hepatic genes and proteins, such as α-fetoprotein (AFP)，albumin (ALB)，glucose-6-phosphatase (G6P)，tyrosineaminotransferase (TAT)，cytokeratin 8 (CK8), cytokeratin 18 (CK18) and urea, to analyse the hepatic differentiation. At last, the hepatic differentiation ratio was also determined by counting the ICC-positive cells. In results, the mRNA of AFP, ALB, G6P and TAT didnt respectively expressed till at day3, day9, day11 and day13. AFP, ALB and urea in culture medium were not detected till at day8, day11 and day12 with a concentration of (3.4±0.6) μg/L, (0.21±0.04) mg/L and (8.3±1.4) μmol/L. Hepatic proteins such as AFP, ALB, CK8, CK18 didnt respectively expressed in cytoplasm till at day7, day9, day9, day11 and ICC-positive cells had morphological structures consistent with mouse primary hepatocytes. Hepatic differentiation ratio was determined at day19 with ratio of 32%. In conclusions, hepatocytes could be obtained in ES cells differentiation system and get matured with differentiation ratio of 32% when growth factors such as TGF, bFGF and HGF added into culture medium. This may produce a new resource of hepatocytes in liver engineering and hepatocytes transplantation for treating hepatic failure.

Abstract:Human ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with molecular mass of 50 ku. RI can inhibit the activity of ribonuclease A (RNase A). Angiogenin (Ang) is a member of RNase A superfamily. RI also can inhibit Ang activities by tight combination. Angiogenesis is an essential condition for the development of tumors and their metastatic dissemination. So anti-angiogenesis will be an efficient method in the inhibition of the growth and metastasis of tumor. The experiment demonstrated that RI might effectively block the angiogenesis that was induced by angiogenin. RI is constructed almost entirely of leucine-rich repeats that might involve in other unclear biological effect. In order to understand further the potential function of RI and investigate the role of RI in invasion and metastasis. The study established a transfection of human RI cDNA into B16 melanoma cells by the retroviral packaging cell line PA317 carrying the pLNCX-RI in vitro. Transfected B16 cells by PA317 carrying the pLNCX and untransfected B16 cells were used as control. The B16pLNCX-RI cell line with a stably high expression of RI was identified by PCR, RT-PCR, Western blot and immunofluorescence assay respectively. The results showed that the transfected RI gene might significantly inhibit cell proliferation, migration, and enhance cell adhesion, as well as, make morphological changes in vitro. Cell doubling time were (24.98±0.16)h, (25.62±0.28)h, (32.64±1.11)h in B16 cells, B16 pLNCX and B16 pLNCX-RI cells respectively. Cell adhesion rate was significantly increased by 19.5% and 17.8% as well as cell migration was reduced by 60% and 61.4% in B16 pLNCX-RI cells compared with pLNCX B16 cells and B16 cells respectively. B16 pLNCX-RI cells became flatter, less nucleoli, less division phases and weaker alkalophilic quality of cytoplasm compared with control groups, which should imply that cell proliferation viability was decreased and malignant phenotype was improved on the cell transfected RI. Mice injected with B16 pLNCX-RI cells show a significant inhibition of the metastasis of tumor with lighter lung weight, fewer metastasis nodules, a lower incidence rate, a lower density of blood vessels and longer survival with respect to the control groups, which implied that RI might be involved in metastasis of melanoma. The results of experiments show RI has a significant antitumor metastasis effect and suggest that it is partially responsible for inhibiting angiogenesis, decreasing cell proliferation, reducing cell migration and enhancing cell adhesion.

Abstract:Neurofibrillary tangles are the neuropathological hallmarks of Alzheimer disease (AD). Abnormally hyperphosphorylated tau and neurofilament (NF) are the components of neurofibrillary tangles. Hyperphosphorylation may be the result of an imbalanced regulation between protein kinases and protein phosphatases. Among the many kinases, glycogen synthase kinase-3(GSK-3) might be a key participator in neurodegeneration of AD. To investigate the role of GSK-3 on Alzheimer-like neurofibrillary degeneration, the wild type mouse neuroblastoma cell lines (N2awt) were treated with wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3K), and the effect of WT on cell metabolism, cell morphology, cell apoptosis, phosphorylation of NF and tau were detected, as well as the relationship between the alternations of these parameters and GSK-3 activity. It was found (1) that treatment of the cell with 1 μmol/L WT led to a transient (at 1h) activation of GSK-3 with a concurrent increase in phosphorylation of NF and tau. At 3h, the activity of GSK-3 was decreased and the hyperphosphorylation of NF was partially restored. (2) that WT decreased the cell metabolism detected by MTT assay in a dose dependent manner. (3) that treatment of the cell with 1 μmol/L WT for 1h or for 3h induced retraction of cell processes. (4) that no typical apoptotic damage was seen by transient stimulation of GSK-3 activity. It is suggested that transit overactivation of GSK-3 led to Alzheimer-like hyperphosphorylation of cytoskeleton protein and impairment in cell viability.

Abstract:To construct recombinant adenovirus vector contained human pancreatic duodenal homeobox-1 (PDX-1) so as to study the expression and effect of PDX-1 gene in bone marrow mesenchymal stem cells, human PDX-1 gene was ligated into shuttle vector pAdTrack-CMV. Homologous recombination was performed in BJ5183 bacteria by cotransforming linearized shuttle plasmid with adenovirus backbone plasmid pAdEasy-1.The recombinant plasmid was packaged and amplified in 293 cells.Mesenchymal stem cells(MSCs) were isolated from healthy adult bone marrow.The adenovirus was transfected into MSCs. The recombinant adenovirus vector has been successfully constructed according to the results of sequencing,PCR and enzyme digestion identification.The expression of PDX-1 and insulin in transfected MSCs at 7 days was detected by RT-PCR and immunocytochemistry. The value of insulin secretion was (15.21±3.50 )mIU/L from the transfected cells at 7days.These results showed that PDX-1 gene modified MSCs could be the beta cell replacement for those diabetes patients who need insulin-secreting cells.

Abstract:To establish a new animal model of the human type 2 diabetes mellitus and investigate the change of ATP binding cassette transporter A1 expression in diabetic minipigs, Chinese minipigs were fed a normal control diet (CD) or a high fat/high sucrose diet (HFSD) for 6 months. Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and glucose were determined by commercially enzymatic methods. Plasma free fatty acids (FFA) were measured by a colorimetric method. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chaim reaction(RT-PCR), Western blot and immunohistochemistry, respectively. After HFSD for 6 months, plasma serum total cholesterol, HDL cholesterol, FFA, triglyceride, and glucose in diabetic minipigs were increased compared with the control. Insulin level of diabetic minipigs was increased in the first 3 months and then decreased at the end of the 6th month. ATP binding cassette transporter A1 expression and liver X receptor α in diabetic minipigs was upregulated. HFSD may induce hyperglycemia, hypercholesterolemia, hypertriglyceridemia and upregulation of ATP binding cassette transporter A1 expression and liver X receptor α in diabetic minipigs.

Abstract:Huwentoxin-Ⅴ (HWTX-Ⅴ) is an insecticidal peptide purified from the venom of spider Selnocosmia huwena. HWTX-Ⅴ contains 35 amino acid residues, including six cystein residues that form three pairs of disulfide bond. The positions of the disulfide bonds of HWTX-Ⅴ have been determined. To map the disulfide bonds, native HWTX-Ⅴ was multi-proteolytically digested. The resulting peptides were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry, and it indicated the presence of one disulfide bond Cys9-Cys21. The partially reduced peptides by using Tris- (2-carboxyethyl)-phosphine at pH 3.0 over 12 minutes were purified by reverse phase high-performance liquid chromatography, and then one -free thiod and two-free thiod fractions were collected. The free thiods were carboxamidomethlate by iodoacetamide at the concentration of 0.5mol/L.The locations of disulfide bond Cys2-Cys16 and Cys15-Cys28 were proved by comparing N-terminal sequencing analysis these partially reduced and alkylated HWTX-Ⅴ with that of the intact peptide. Finally, the three disulfide linkage of HWTX-Ⅴ could be assigned as Cys2-Cys16,Cys9-Cys21 and Cys15-Cys28.

Abstract:In order to elucidate the location of chorismate mutase and prephenate dehydrogenase on E.coli T-protein, protein limited digestion and fragmentation cloning were employed. Fragment 1～93 and fragment 96～373 were cloned and expressed according to the results of limited digestion of T-protein respectively. Two fragments were found to have chorismate mutase and prephenate dehydrogenase activities respectively. In a conclusion, E.coli T-protein do have independent domains, N-terminal 93 amino acids belongs to chorismate mutase and C-terminal 277 amino acids belongs to prephenate dehydrogenase.

Abstract:One of the fundamental changes during apoptosis is the abnormality of cytoskeleton proteins, which determines some morphological features of apoptotic cells. To understand the role of apoptotic proteases, granzyme B and caspase-3, in the cleavage of the cytoplasmic form of actin, an alternative cell free system based on adult monkey brain tissue was used to reproduce the downstream part of apoptotic program, initiated by the addition of granzyme B. Through extensive Western blot analyses, it was showed that β-actin was cleaved to 41 and 15 ku fragments in the granzyme B-treated brain extract after a 12-hour incubation. The production of these two fragments was further found to be granzyme B dependent. Neither endogenous caspase-3 activated by granzyme B nor its recombinant active form was capable of processing the actin in the brain extract, although the enzyme cleaved the actin purified from rabbit skeleton muscle to produce the 15 ku fragment. The results suggest that endogenous β-actin is resistant to apoptotic proteases, especially to caspase-3, either because of conformational constrains between actin and these proteases or due to the presence of other factors that prevent degradation.

Abstract:An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantitate the Klebsiella pneumoniae (Kp) capsule glycoprotein. The rabbit antiserum of Klebsiella pneumoniae capsule glycoprotein was prepared successfully by using a purified Kp capsule glycoprotein as antigen and the antibody was purified with ammonium sulfate fractionation and adsorption. The sensitivity of the assay is 0.031 mg/L. The standard curve is linear from 2.5 mg/L to 30 mg/L. The error of inter-plate is from 1.04% to 3.22%, and the error of intra-plate is from 2.61% to 8.35%.

Abstract:The molecular diagnosis for X-linked adrenoleukodystrophy (ALD) using mutational analysis at genomic DNA level is important. However, some ABCD1 gene mutations were difficult to detect by conventional methods, such as PCR-RFLP and the direct DNA sequencing of PCR product, because of the interference of the pseudogenes. To avoid the interference, genomic DNA from the family members with an adrenoleukodystrophy gene mutation(R617G mutation) was analyzed by amplification refractory mutation system. The results indicated that amplification refractory mutation system is one of the effective methods for avoiding the interference of the pseudogenes in detecting ABCD1 gene mutations.