Abstract:RNAi is an efficient antiviral system, and viral gene-specific siRNAs are very promising antiviral inhibitors. However, many viruses have evolved highly sophisticated mechanisms that interfere with both siRNA- and miRNA-guided silencing pathways. Deeper understanding the strategies exploited by viruses provides the basis for the development of effective RNAi-based therapies that prevent viral evading. Therefore, the latest progress on the strategies exploited by viruses for evading the RNAi is reviewed.

Abstract:LRRC4, a novel member of LRR (leucine-rich repeat) superfamily, is cloned by expressed sequence tag (EST)-mediated positional cloning strategy combined with 5′-RACE technology. Normal expression of LRRC4 is highly specific for brain, whereas absent or significantly down-regulated in primary tumors including glioma, meningioma and pituitary adenoma. LRRC4 is a functional gene in neural development and axon growth, and associated with glioma grade progression. LRRC4 expression is gradually reduced, even absent accompany with glioma grade increase. Absent expression of LRRC4 is involved in the late event of malignant glioma progression.The reexpression of LRRC4 can decrease a series of growth factors/neurotrophic factors (IGF, EGF, PDGF, CNTF, bFGF,GDNF and BDNF) or receptors gene expression to regulate RTK-mediated many signaling transduction pathway, such as K-Ras/c-Raf/ERK/MAPK, PI-3K/AKT/NF-κB, p70S6/PKC, STAT3 and JNK2/c-Jun/mp53, which block U251 cells in late phase of G1 to inhibit glioma cells proliferation and invasion. This inhibitory effect of LRRC4 is dependent on its LRR domain. LRRC4 induces glioma cells to differentiate into astrocyte-like cells more than apoptosis.

Abstract:Inhibitor of growth (ING) family proteins belong to candidate tumor suppressor proteins. The ING proteins participate in PtdInsPs-mediated lipid signaling and hormone signaling pathways. They are associated with histone acetyltransferase, histone deacetylase and play a role in chromatin remodeling and gene transcription regulation. ING proteins regulate cell growth, apoptosis and DNA damage repair in p53 dependent manner; thus linking the processes of cell cycle regulation, apoptosis and cellular aging through epigenetic regulation of gene expression.

Abstract:It is widely accepted that RNA silencing or RNA interference plays a crucial role in defending viruses from invasion and regulating gene expression, and has become a very potent tool for genetic function studies and some disease therapies. RNA silencing ubiquitously exists in eukaryotic cells, however, during the boundless process of synergistic evolution for the host and viruses, viruses have gained capacities of escaping or suppressing RNA silencing to make RNA interference sterile. On the other hand, some studies have revealed that mammiferous cells themselves also could regulate the effects of RNA silencing, which presents the regulation of vital activities under a more perfect condition. In order to develop the potential function of RNA silencing, people have worked out a number of strategies to decrease the suppression effects of RNA silencing. Here, all kinds of suppress mechanisms and application about RNA silencing were summarized in order to make people take cognizance of the disadvantage of RNA silencing technology while using it.

Abstract:Eukaryotic genomes are composed of individual genes and their intergenic regions. Traditionally, it has been held that the transcription of a gene always begins from the transcription start site and ends at the termination site, the gene is referred as an individual transcription unit. However, some sporadic studies indicate that transcription can sometimes read-through the intergenic region and generates a large fusion transcript which contains the upstream gene, intergenic region and the downstream adjacent gene. The fusion transcript becomes a mature functional transcript after intergenic splicing. The patterns of intergenic splicing, possible generation mechanism and its significance are summarized.

Abstract:Embryos that were produced by parthenogenesis were cultured to morula, then each cell of morulas was transferred into enucleate MⅡ oocytes, as called parthenogenetic morula nuclear transplantation embryos here. NIH3T3 cell nucleus were also transferred into enucleate MⅡ oocytes. In order to prove up the relationship between nuclear reprogram and DNA methylation during the process of nuclear transplantation, the two cloned pre-implantation embryos together with in vitro fertilization (IVF) and parthenogenetic embryos were subjected to immunocytochemistry with antibody to DNA methylation (5'-MeC). In order to research the regulation of oocyte cytoplasm to the relative expression level of imprint genes, the relative expression level of U2afbp-rs gene which is maternally imprint and non-imprint eIF-4C gene in pre-implantation IVF, parthenogenetic and parthenogenetic morula nuclear transplantation embryos by real-time PCR were detected.The results show that neither the genomic DNA of NIH3T3 cloned embryos nor parthenogenetic morula nuclear transplantation embryos were demethylated actively. Although both the relative expression level of U2afbp-rs gene and eIF-4C gene in parthenogenetic morula nuclear transplantation embryos were lower than control parthenogenetic embryos, the expression of the two genes were similar to the control parthenogenesis group. These results suggest that the imprinting gene expression of donor nuclear are regulated by oocyte cytoplasm.

Abstract:In four patients with chronic pancreatitis from two hereditary pancreatitis (HP) families and 63 normal controls, five exons of cationic trypsinogen gene (PRSS1) were amplified by PCR and it?蒺s products were analyzed by sequencing, related clinical data were also collected. All the four patients were found mutations in the PRSS1 gene but their clinical feature is absolutely different. Six patients with diabetes mellitus were found in pedigree No. 1, it's members show pancreatitis symptom later, at about 29, the tumor markers (CA19-9, CA72-4) is obviously higher than the patients in pedigree No. 2, two patients with chronic pancreatitis in pedigree No. 2, show symptom earlier without diabetes mellitus, their clinical characterization are different too. The number of CD4⁺T cell/ CD8⁺T is very low in Ⅲ8, but Ⅲ7 is normal, and the level of anti-HBs of Ⅲ8 is variable in the course of pancreatitis, but the phenomenon was not found in Ⅲ7. In their PRSS1 gene two guanosine (G) to adenosine (A) mutations were found in PRSS1 exon 3 of pedigree No. 1, one was detected at 336 basyl, the other mutation occurs at 361 basyl. The results of the mutations were Lys →Lys and Ala →Thr. While thymine (T) to adenosine (A) and (guanosine) G→(adenosine) A mutation in PRSS1 exon 3 was detected in the other patient of pedigree No. 2 (Ⅲ8). One was 361 basyl, the other at 415 basyl. While c.415 T→A was not found in the proband of pedigree No. 2 PRSS1 gene (Ⅲ7). All of the mutations were heterozygous mutation, that is to say all of the trypsinogen were wild type and mutant type concomitance, the normal and abnormal pathway of active trypsinogen exist partially. At the same time, the mutations of SPINK1 were not observed. Compared with the documents and registration of NCBI, it can be concluded that PRSS1 gene had many kinds of mutations in hereditary pancreatitis, the heterozygous mutations (c.336 G→A, c.415 T→A) were the novel mutations and related with clinical phenotype. What's more, it's the first time that the multisite heterozygous mutations of PRSS1 gene were reported. The presence of the mutations in four patients with chronic pancreatitis, it's absence in their relatives and the strong evolutionary conservation of the mutation, all indicate that the trypsinogen mutation is associated with hereditary pancreatitis and for the first time raises the question whether a gain or a loss of trypsin function participates in the onset of Chinese pancreatitis.

Abstract:34 captive Giant Pandas (captive population: including a population and b population) and 7 captive wild Giant Pandas (captive wild population) are study objects. Their blood samples are got from Chengdu Research Base of Giant Panda Breeding and China Research and Conservation Center for the Giant Panda. 30 microsatellite DNA markers including AY161177～AY161218, Ame-μ5～Ame-μ70 and g001～g905 are used to investigate the actual state of genetic diversity within and between samples. Meanwhile, the measures how to maintenance and improve genetic diversity of Giant Pandas are discussed. The information for microsatellite locus showed that 30 microsatellite DNA markers were polymorphic (PIC=0.621～0.640) and the genetic diversity of 41 captive Giant Pandas was higher (a population: A=5.48, Ho=0.475, He=0.690; b population: A=5.24, Ho=0.453, He=0.719; captive wild population: A=3.80, Ho=0.514, He=0.725) than that of other 6 endangered species(Ho=0.210～0.390, He=0.150～0.430), but was lower than that of 3 non-endangered species. 41 captive Giant Pandas maintenanced high genetic diversity. However, compared to 7 captive wild Giant Pandas, the genetic diversity of 34 captive Giant Pandas degraded. The date of F-statistics and gene flow (of 25 microsatellite locus Nm=2.610, Fst=0.0874, Fit= 0.4116) indicated that a population and b population exchanged individuals resulting to inbreeding. There was a low level of genetic variabilities between a population and b population. The inbreeding level of b population was higher than that of a population (a population: Fis=0.3221，b population: Fis=0.3983). Therefore, at the present, the focus of management of captive Giant Panda should shift to avoiding the inbreeding and the loss of genetic diversity. The captive populations of Giant Panda should be in the same management unit. Retrieving pedigree and chosing the fittest exchange individual are the key point. Microsatellite technologey is the key way to protect and improve genetic diversity of captive Giant Pandas.

Abstract:The effects of atomic oxygen radical anion (O^－) on the inactivation and morphological changes of Escherichia coli (E.coli) on the surface of bio-indicator carrier were investigated. The O^－ flux was generated from a novel developed O^－ generator where the O^－ storage-emission material of [Ca₂₄Al₂₈O₆₄]⁴⁺·4O^－ (abbreviated as C12A7-O^－) was used as a pure O^－ emitter. Present results showed that inactivation of E.coli was sensitive to the O^－ intensity and the cell mortality was enhanced to more than 3-logarithm reduction with the exposure to 1.5 mA/cm² O^－ flux for 120 min. Field emission scanning electron microscopy (FESEM) observations showed that O^－ flux destroyed cellular structures. Lipid peroxidation reaction induced by atomic oxygen radical anion for E. coli cells was also observed using product of malondialdehyde (MDA) as an index. The concentration of MDA increased to 1.2 μmol/g(dry weight) of cells when E. coli suspension (5.6×10⁷ cfu/ml) was treated by the O^－ flux (1.5 μA/cm²) for 15 min. The findings revealed that the atomic oxygen radical anions, with strong oxidation power, was effective in inactivating E. coli and caused lipid peroxidation reaction at the first time, which would be potential useful to develop a novel approach for the microbial decontamination and for the study on the interactions between microorganisms and O^－.

Abstract:A proteomics approach was applied to analyze differential expression of proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient (IPG, ranges 4～7) strips was used to separate proteins. On the 2-DE gels stained by Coomassie brilliant blue, PDQuest image software detected about 550 protein spots, of which 10 spots show more than 2.5-fold changes in abundance between wild and cultivated soybeans. These 10 proteins treated by tryptic in-gel digestion were characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and peptide mass finger printings of all were obtained. Soybean UniGene database and NCBI were used to identify proteins, of which 5 proteins were identified. They were soybean agglutinin, seed maturation protein PM24, maturation polypeptide, sucrose-binding protein precursor and trypsin inhibitor p20. The potential functions of these identified proteins were discussed.

Abstract:The Na⁺/H⁺ antiporter in vacuolar membranes transports Na⁺ from the cytoplasm to vacuoles using a pH gradient generated by proton pumps, which could reduce Na⁺ toxicity. It is uncertain that whether the woody plants have the same mechanism. Through differential centrifugation and sucrose density gradient centrifugation, tonoplast vesicles were isolated from Populus tremula calli broken by blender. After establishing pH gradient by V-ATPase, Na⁺ could dissipate the pH gradient, which indicates that there is Na⁺/H⁺ antiporter in the tonoplast vesicles from Populus tremula calli (K_m=11.4 mmol/L). Amiloride could inhibit the Na⁺/H⁺ antiporter activity. The antiporter could transport Na⁺ and K⁺, the affinity for Na⁺ is higher. Salt stress decreased K_m and V_max.

Abstract:Cyclin-dependent kinase 5(Cdk-5) and protein kinase A(PKA) are the important kinases in the regulation of Tau phosphorylation. However, it is unclear weather they participate in the regulation of tau phosphorylation in diabetic rat brain. The roles of Cdk-5 and PKA in the regulation of abnormal tau hyperphosphorylation in the hippocampus of diabetic rat model have been investigated. The diabetic rat model was induced by intra peritoneal injection with 55 mg/kg streptozotocin. Intracellular free calcium concentration was detected by loading Fura-2 and fluorescent technique in Control, Con+ Ros, DM, and DM+Ros groups, and the activities of Cdk-5 and PKA were measured by immunoprecipitation and by liquid scintillation for incorporated radioactivity respectively, furthermore, Western blotting was used to examine the level of tau phosphorylation by using phosphorylation-dependent and site-specific tau antibodies. The results show that, compared to normal control, the concentration of intracellular calcium in the diabetic rat hippocampus was elevated to 165.92 nmol/L(P < 0.05). Cdk-5 activity was increased by 29% of control, and an increase of PKA activity by 30% of control was also found, both Cdk-5 activity and PKA activity were increased highly compared to normal control(P < 0.01, P < 0.01), furthermore, the immunoreactivity of Tau-1(detection of Ser 198/Ser 199/ Ser202, non-phosphorylated site) was decreased(P < 0.01), the immunoreactivity of PHF-1 sites (detection of ser396/ser404, phosphorylated site) was increased(P < 0.05) in these diabetic rats. After treated the DM rats with roscovitine, a specific Cdk-5 inhibitor, Cdk-5 activity was inhibited to 105% vs control, it was not obviously increased compared to control (P >0.05), but PKA activity remained 127% vs control, it was high compared to control(P < 0.01). Moreover, the hyperphosphorylation of tau at Ser198/Ser199/Ser202 epitopes was reversed (P < 0.01), but the level of phosphorylation of tau at Ser396/Ser404 epitopes was remained highly after roscovitine was administered to DM rats. However, after treated the control rats with roscovitine, Cdk-5 activity was about 104% vs control, and PKA activity was 105% vs control, neither Cdk-5 activity nor PKA activity was higher compared to control(P > 0.05), and the level of tau phosphorylation at Ser198/Ser199/Ser202 and Ser396/Ser404 sites was not increased compared to control too(P > 0.05). These results firstly suggest that the increase of Cdk-5 activity and PKA activity may cause the hyperphosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 epitopes in diabetic rat hippocampus, and the intracellular calcium may play a role in this process, however, it is needed to be elucidated in great details.

Abstract:The different effects of cyclosporine and rapamycin on tumor progress and metastasis and their mechanisms were investigated. BTT-T739-gfp cell line stably expressed green fluorescence protein was intracutaneously inoculated into 24 mice. One week later, the mice were randomly divided into 3 groups and treated intraperitoneally by normal saline (as control), cyclosporine and rapamycin respectively. Survival rate and tumor volume were measured. The tumor metastasis, pathological angiogenesis and expression of angiogenesis-associated genes were analyzed. The metastasis analysis in lung and liver were also accomplished under the fluorescent microscope. Compared with the normal saline and cyclosporine, the general immunosuppressive dosage of rapamycin effectively inhibited the progress and metastasis of the established tumors. It was the rapamycin that improved the survival rate of tumor-beared mice and decreased the developmental velocity of tumor volume, to which was significantly different from the control and cyclosporine groups on the 12th day and 14th day. Experimentally, rapamycin inhibited tumor growth through angiogenesis and metastases repression in the established mouse model. From a mechanistic perspective, rapamycin showed anti-angiogenetic activities related to decrease of VEGF-A production which was a result of the down-regulation on the transcriptional level of VEGF-A and its transcription activator HIF-1α. It implied that conventional immunosuppressants showed different therapeutic roles on established tumors. Rapamycin, not cyclosporine could inhibit the tumor growth and metastasis of which was referred to anti-angiogenesis.

Abstract:Thymidylate synthase (TS), an essential enzyme for DNA de novo synthesis, is a critical therapeutic target in cancer therapy. Previous study has shown that TS was able to bind to its own mRNA in human and E.coli, resulting in translational repression. Zebrafish is the best animal model for vertebrate study. In order to study the regulatory mechanism of zebrafish TS, the enzyme were expressed in E. coli BL21 (DE3) and it was purified to homogeneity. Electrophoretic mobility shift assay (EMSA) was used to detect the interaction of zebrafish TS protein and its own TS transcript in vitro and the results showed that zebrafish TS could bound with its own mRNA specifically. Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunoprecipitation : RT-PCR technique. The results provide evidence that zebrafish may be developed as an useful model for studying the anti-metabolism agents.