Abstract:Homo sapiens PDCD10 (programmed cell death 10, alias, “TF-1 cell apoptosis related gene 15, TFAR15”), cloned by means of cDNA-representational differences analysis, had been initially identified associated with cell apoptosis. Recent research suggested mutations within the PDCD10 gene or deletion were responsible for cerebral cavernous malformations, and PDCD10 was the third CCM gene. On the other hand, other research demonstrated that PDCD10 was strictly modulated and up regulated in many kinds of tumors, which implicated that PDCD10 participated in tumorous signal transduction. The recent research confirmed that PDCD10 interacts with MST4, a member of Ste20-related kinases, and the interaction promoted cell proliferation and transformation via modulation of the ERK-MARK pathway. In conclusion, all these demonstrate that PDCD10 has many biological effects, which suggests that it is a novel player in vascular morphogenesis and/or remodeling, as well as tumorigenesis and cancer progression.

Abstract:The combination of tandem spectrometry and database searching is one of the most popular technologies for protein identification. However, only those proteins in the searching database could be identified, and current database is far from completeness. So it is necessary to mining the MS/MS data comprehensively, in which novel protein identification is the most important one. The definition of novel protein could be divided into three levels according to their annotations of sequences and functions. As a part of protein identification, the main approaches used to identify novel protein are basing on the following two different ways: de novo sequencing combined with similarity search and searching against nucleotide acid databases such as EST or genome databases. Several mature or newly developed methods and techniques were summarized, and the problems and strategies discussed here would be helpful for the related researches.

Abstract:UCH-L1 (ubiquitin carboxy-terminal hydrolases L1) is a member of the carboxyl-terminal ubiquitin hydrolase family, naturally, that there is hydrolase activity, however, and that there is ligase acitivity, which is different from the other members of UCHs. UCH-L1 hydrolase activity could keep the pool of free ubiquitin and compromise the ubiquitin-dependent degradation pathway, while UCH-L1 dimerizational-dependent, ubiquityl ligase activity could produce undegradable, K63-linked polyubiquitin chains that could inhibit proteasomal activity. Therefore, UCH-L1 is involved in the more diverse physiological activities, including neuron formation, gonadal development and fertilization. Mutation of UCH-L1 is linked to the neurodegenerative diseases, such as Parkinson's disease. Moreover, abnormal expression of UCH-L1 is response to carcinogenesis in many tissue such as thyroid lung.

Abstract:RNA interference (RNAi) is a gene-silencing progress induced by double stranded RNA at a level of posttranscription. At present, RNAi has been extensively applied to the research domain of gene functions and disease therapies, especially for the therapy of malignant tumours due to its simple designs, immediate effects and obvious efficiency. Up to now, a number of novel strategies have been engineered to effectively fight against malignancies through RNAi technology in the regulation of the tumorigenesis-and-progression-associated genes. Presently, uncontrolled RNA Pol Ⅲ promoter expressing classical small hairpin RNA which can be processed into siRNA by Dicer enzyme is extensively applied. However, most of the current methods lack of tumor targeting and high efficiency in cancer therapy. The latest studies have demonstrated that RNAi induced by the tissue specific RNA Pol Ⅱ promoter could compensate for a deficiency of RNAi mediated by RNA Pol Ⅲ promoter. Moreover, virus vectors, especially cancer-specific replicable adenovirus targeting to cancer cells and oncolysising, which can express siRNA controlled by RNA Pol Ⅱ promoter, is expected to be a more effective therapy strategy.

Abstract:To clarify the gene and molecule characters of the tetrodotoxin-resistant (TTX-R) voltage -gate sodium channel and the distribution in different lobe of brain in different developmental stage, reverse transcriptase- polymerase chain reaction (RT-PCR) was used to clone the full sequence of Nav1.5 sodium channel (designated as rN1) α subunit in rat brains and the distribution was compared in different lobe of brain in different developmental stage. The open reading frame(ORF) of rN1 encodes 2016 amino acid residues and sequence analysis indicated that rN1 is highly homologous with 96.53% amino acids identity to rat cardiac Nav1.5 sodium channel (rH1) and 96.13% amino acids identity to human neuroblastoma Nav1.5 sodium channel (hNbR1). It has all the structural features of a voltage-gated sodium channel and the presence of a cysteine residue (C373) in the pore loop region of domain Ⅰ suggests that this channel is TTX resistant. A new exon (exon7) that distinct from rN1 was found in DⅠ-S3～S4. In addition, an alternative splicing isoforms that deleted 53 amino acids(exon20) was found in the loop between DⅡ～Ⅲ in rN1(designated as rN1-2). Distribution result demonstrated that rN1 expressed discrepancy in different ages and lobe in brain. The expression level of rN1 was gradually more stable in adult than neonatal. These results suggest that Nav1.5 has a newly identified exon for alternative splicing and is more widely expressed than previously thought.

Abstract:Full length cDNAs of Cucumber mosaic virus (CMV) CB7 strain, causing necrosis on Nicotiana glutinosa, were obtained by RT-PCR, using viral genomic RNAs as templates. cDNAs of CMV-CB7 genomic RNAs were cloned and sequenced and results indicated that RNA1, 2 and 3 was 3 356 nt, 3 045 nt and 2 218 nt, respectively (accordingly Accession Number EF216866, DQ785470 and EF216867). Infectious RNA transcripts from cDNA clones of CMV-CB7 were inoculated onto N. glutinosa and the seedlings of host plants displayed necrosis symptom, whist that of CMV-Fny induced typical mosaic symptoms. Through pseudorecombination between CMV-CB7 and CMV-Fny genomic RNAs, the genetic determinant of necrosis phenotype was mapped to RNA2. Chimeric infectious clones consisting of partial sequences of RNA2 derived from CMV-CB7 and CMV-Fny, respectively, were obtained by Overlapping PCR. Pathogenic analysis with those chimeric RNA2 revealed that 2b gene or 3′UTR of CMV-CB7 RNA2 was responsible for the necrotic pathotype. Northern blotting analysis reflected that both necrotic and non-necrotic viruses accumulated to similar levels of genomic RNAs in host plants. Therefore, necrotic phenotype induced on N. glutinosa was not related to the level of accumulation of CMV genomic RNAs.

Abstract:In order to study the feasibility and transfection mechanisms of a novel gene vector system composed of plasmid DNA-anti DNA antibody-cationic lipid (DAC) for non-virus site-specific gene therapy, stable DAC triplex nano-micelles were formed in PBS system through self-assembling of plasmid DNA, anti DNA antibody and cationic lipid. The mechanisms of cell uptake and entry with DAC were observed by fluorescent confocal microscopy. Transfection efficiency of DAC was estimated by using A10 cell in vitro with GFP-DNA (green fluorescent) triplex micelles. Cell culture studies revealed that DAC triplex micelles markedly increased the level of gene transferred to A10 cells, with more than 4-fold increase in transfection compared to DC group without anti DNA antibody and 11-fold increase compared to DA group without cationic lipid. Fluorescent DNA studies demonstrated greater cell uptake in vitro with DAC compared to the control formulations. Confocal microscopy studies confirmed nuclear entry in A10 cells with DAC, while formulations without anti-DNA antibody demonstrated no nuclear entry. It can be concluded that plasmid DNA-anti DNA antibody-cationic lipid (DAC) triplex micelle was a novel non-viral gene vector with high transfection efficiency and no cytotoxicity. It was hypothesized that DAC triplex micelles could enhance DNA delivery through mechanisms involving: increased levels of DNA incorporation into micelles due to antibody binding, enhanced plasmid DNA entry into nuclear due to the nuclear entry properties of anti-DNA antibody, and increased transfection efficiency via co-operation of anti-DNA antibody and cationic lipid. Gene delivery using DAC triplex micelles should be suitable for a wide array of single or multiple therapeutic gene strategies and for further successful gene therapy.

Abstract:Efficient gene delivery and sustained gene expression are required for successful human gene therapy. Although viral vectors are considered the most efficient vehicles for gene transfer, currently available viral vectors have not fully achieved these two requirements. Lentiviral vectors (LVs) can integrate into host chromosomes, allowing long-term gene expression, in addition, these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome , but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments. In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks, a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelope plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000^TM. After 4 days, the culture supernatant of lentiviral vectors was collected, the RNA from the supernatant was examined by the RT-PCR, the protein from the supernatant was examined by Western blot, and the supernatant was used to transfect normal 293T , HepG2 and Vero, which were observed by the immunofluorescence microscopy. The type of cell lines, plasmids dosage and the MOI (the proportion between cell numbers and virus copies) were considered so critical to the output of this system that 3×3×3 factorial design was used to explore the yield optimization of this system. As judged by the results of RT-PCR and the Western blot, lentiviral vectors were found in the culture supernatant; as judged by immunofluorescence with microscopy, 293T, HepG2 and Vero which were transfected by the supnantant expressed the report protein - green fluorescent protein(GFP), the results confirmed the valid infectivity of the lentiviral vector produced by the system. Eventually, the best titers of lentiviral vector stocks was up to 1.3×10⁸ tu/ml, which is one order of magnitude higher than the output of classical manufacture system. The new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.

Abstract:FabB (β- ketoacyl-acyl carrier protein synthase Ⅰ) and FabF(β- ketoacyl-acyl carrier protein synthase Ⅱ) are two key enzymes of fatty acid biosynthesis in E.coli. The Gram-positive pathogenic bacterium Enterococcus faecalis has a fatty acid composition very similar to that of E.coli. Bioinformatic analysis reveals that though E. faecalis has two fabF homologues, there is no recognizable fabB homologue in the genome of E. faecalis. Two fabF homologues (fabF1 and fabF2) were amplified by using E. faecalis V583 genomitic DNA as template, and two plasmids, pHW13 (fabF1) and pHW14 (fabF2), were constructed. The results of experiments in vivo and in vitro have shown that fabF1 gene could complement E. coli fabB mutation and FabF1 possessed β- ketoacyl-acyl carrier protein synthase Ⅰ(FabB) activity, while fabF2 gene could complement E. coli fabF mutation and FabF2 had β- ketoacyl-acyl carrier protein synthase Ⅱ(FabF) activity. Meanwhile the data also shown that FabF2 possessed partial function of β-ketoacyl-acyl carrier protein synthase Ⅰ(FabB), and it could make E. coli fabB mutation synthesized low amount of unsaturated fatty acid. From these data it is clear that FabF species enzymes could have activity of β- ketoacyl-acyl carrier protein synthase Ⅰ(FabB).

Abstract:In order to study the changes of main oxidase and antioxidase in the pathological scars, the tissues of hypertrophic scar(10 cases), keloid(10 cases) and normal skin(8 cases)were obtained. The concentration of malonaldehyde (MDA) and the activities of xanthine oxidase(XO), copper, zinc-superoxide dismutase(CuZn-SOD)，catalase(CAT) as well as glutathione peroxidase(GPX) were detected by spectrophotometric method. Compared with normal skin tissues, the concentration of malonaldehyde and xanthine oxidase activity were significantly higer in pathological scars (P < 0.05), SOD activity were higher, CAT activity was decreased remarkably in pathological scars than that in normal skin tissues (P < 0.05). And the activity ratios of CAT/CuZn-SOD and GPX/CuZn-SOD were obviously reduced (P < 0.05). However, no significantly differences were observed in the decrease activity of GPX. Differences in above-mentioned indexes all were not remarkable between hypertrophic scars and keloids. These data suggest that the changes of XO, CuZn-SOD, CAT and GPX activities may be a reason resulted in an increase in the level of reactive oxygen species in pathological scars. In the alternative of antioxidants, CAT may be more reasonable.

Abstract:Lymphatic metastasis is the first step in the metastatic process of magligant tumors deriving from epithelia, and its uncertain mechanisms are always the major problem in the field of oncology. In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F with highly lymphatic metastatic potentiality and Hca-P with low lymphatic metastatic potentiality were compared using fluorescent two-dimensional difference gel electrophoresis(2D DIGE). DeCyde software was applied to analyze 2D DIGE images,and 163 differential protein spots between Hca-F and Hca-P were detected, including 86 up-regulated protein spots in Hca-F and 77 down-regulated protein spots in Hca-F. 23 protein sports representing differential ratio more than 2-fold were chosen to be analyzed by MS. 17 metastasis-associated proteins were identified, and the partial identified proteins were further validated by Western blotting analysis. Of the identified proteins, the expression of transketolase, vimentin, creatine kinase(brain), anxa7 protein, anxa5 protein,enoyl coenzyme A hydratase 1(peroxisomal), heterogeneous nuclear ribonucleoprotein A2/B1 isoform 1 were up-regulated in Hca-F. Whereas eukaryotic translation elongation factor 2, Ero1l, Aldh2 protein, malate dehydrogenase 2(NAD), lactamase beta 2, glutathione S-transferase omega1, ubiquitin carboxyl-terminal hydrolase isozyme L3, endoplasmic reticulum protein ERp29(precursor), lypla1, stathmin were down-regulated in Hca-F. On the basis of the Gene Ontology(GO)classification，the differential expression proteins were found to be involved in many of biological process, such as metabolism, protein secretion, protein binding, nucleic acid binding, calcium ion binding,apoptosis and regulation of growth etc. Validating the function of these proteins is helpful to elucidate the mechanisms of lymphatic metastasis.

Abstract:Obesity and its related metabolic diseases become major health problems in the world. Adipose tissue plays an important role in the development of obesity. FSP27, a member of the CIDE family proteins, is expressed at high levels in white adipose tissue and differentiated 3T3L1 cells. The objective of current study is to establish a FSP27 knockdown preadipocyte cell line to investigate the in vivo function of mouse FSP27. The double strand siRNA of mouse FSP27 corresponding to nucleotides 270 to 291 was synthesized and inserted into pSilencer2.1. pSilencer-siFSP27 was co-transfected into 293T cells with the HA-mFSP27 expression vector to test its knock-down efficiency. The FSP27 siRNA was then transferred to a lentiviral vector. Lentivirus were generated and used to infect 3T3-L1 cells. It was shown here that lentivirus containing FSP27siRNA can effectively knockdown FSP27 expression in 3T3-L1 cells. Establishment of FSP27 knock-down cell line provides a useful tool for the study of in vivo function FSP27.

Abstract:Analysis of cellular pathways and networks in terms of logic relations is important to decipher the networks of molecular interactions that underlie cellular function. A computational approach for identifying lower and higher order gene logic associations was presented on the base of graph coloring theory and applied it to the colon cancer mRNA microarray data. Then the logic relationships of 51 oncogenes and cancer suppressor genes are analyzed and the logic association network of them was constructed. The signal pathway of TGFβ from the network model was found and verified by the colon cancer pathway of KEGG. The model reveals many higher order logic relationships of cancer genes. These relationships illustrate the complexities that arise in cancer cellular networks because of interacting pathways. The results show that this method is feasible and is expected to give a reference to the medical molecular biologist.

Abstract:BRD7 is a novel bromodomain gene isolated by cDNA representational difference analysis (GenBank accession number: AF152604). Ectopic expression of BRD7 inhibited NPC cell growth and cell cycle progression.Previous studies demonstrated that BRD7 gene could regulate the activity of Rb/E2F pathway. In order to further explore the molecular mechanisms of BRD7 regulating Rb/E2F pathway, Westernblot and RT-PCR analysis were carried out. Results showed that BRD7 could inhibit the phosphorylation of Rb, decrease the expression of cyclin D1 and cyclin E, up-regulate p19 in RNA level, but had no effects on the expression of CDK4 and CDK2. Luciferase reporter assay suggested that BRD7 repressed cyclin D1 promoter activity. Furthermore, an antisense nucleic acids technology was performed to silence the endogenous BRD7 gene in COS7 which resulted in the upregulation of cyclin D1, cyclin E, phosphorylated Rb, and acceleration of the cell growth. As a result of this research, BRD7 inhibits G1-S phase progression in cell cycle via regulating the important molecules involved in Rb/E2F pathway.

Abstract:The 2b protein encoded by Cucumber mosaic virus (CMV) plays an important pathogenicity role in many solanaceous hosts, but mechanism of inducing disease is still unknown. In order to investigate virulence of the 2b protein on Nicotiana glutinosa plants, in terms of chloroplast structure and photosynthesis, a mutant Fny-CMVΔ2bpro, which cannot express the 2b protein, was achieved by introducing mutant sites in the 2b gene of Fny-CMV. N. glutinosa seedlings were inoculated with wild-type Fny-CMV and the mutant Fny-CMVΔ2bpro, and were analyzed for symptom expression, chlorophyll content, photosynthetic rate, and ultra-structural alteration of chloroplast. Up to 30 days post inoculation, wild-type Fny-CMV caused symptoms of severe mosaic, leaf deformation, and stunting, reduced photosynthetic rate and chlorophyll content, and altered the ultra-structure and morphological characters of the chloroplasts. However, host seedlings inoculated with the mutant Fny-CMVΔ2bpro expressed only slight mosaic symptom. Their photosynthetic rates and chlorophyll contents were not significantly different from those of the mock-inoculated plants, and the ultra-structure and morphological characters of their chloroplasts appeared to be normal. The observed low photosynthetic rates and chlorophyll contents were related to the breakage of the chloroplast morphology and ultra-structure. Results of Northern blotting showed that the virulence of 2b protein was associated with high accumulation level of CMV progeny RNAs in systemic leaves. Non-expression of the 2b protein reduced the accumulation levels of its genomic RNAs 1 and 2. The level of subgenomic RNA4, encoding CP protein, was found to be significantly decreased.