Abstract:Cell migration plays a pivotal role in many physiological and pathophysiological processes including embryogenesis, angiogenesis, immune defense, wound healing, or the formation of tumor metastases. Evidence is accumulating that ion channels and transporters is also required for optimal cell migration and a model is proposed that combines ion transport with cytoskeletal mechanisms of cell migration. However, the role of water transport in cell migration is underinvestigated. In the April 7, 2005 issue of Nature, Saadoun et al. discovered the importance of aquaporin-mediated transmembrane water flux in cell migration, angiogenesis and tumor growth, provided a new molecular mechanism of cell migration and opened a new field of aquaporin gene function and therapeutic application.

Abstract:Therapeutic cloning has been suggested as a new strategy to treat a number of diseases. Based on the recent breakthrough made by Korean scientists, here the progresses in the field of therapeutic cloning were reviewed and two important issues in this field: the reprogramming after nuclear transfer and the establishment, self-renewal and differentiation of human embryonic stem cells were discussed. The great significance and existing challenges of therapeutic cloning strategy were also prospected.

Abstract:Neuron-restrictive silencer element or repressor element-1 (NRSE/RE1), present in the transcriptional regulatory regions of multiple neuronal-specific genes, is a 21~23 bp conservative DNA sequence. Neuronal restricted silencing factor or RE1-silencing transcription factor (NRSF/REST) can bind to the NRSE, and then the gene repression was mediated in part through the association of its NH2-terminal repression domain with the corepressor mSin3, resulting in the recruitment of histone deacetylase (HDAC) and consequent acetylization, and its COOH-terminal repression domain with the corepressor CoREST, that may serve as a platform protein for assembly of specialized repressor machinery. The recent study show that NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with protein NRSF at transcriptional level, rather than through siRNA or miRNA at posttranscriptional level.

Abstract:Forkhead box (Fox) proteins are transcriptional factors, designated as the unified symbol for all chordate winged helix/forkhead transcription factors by Forkhea/Winged Helix nomenclature committee, and issued in the year of 2000. To date, over 100 forkhead genes have been identified in organisms ranging from yeast to humans. The importance of FOXO subfamily of Fox proteins in the fields of animal lifespan, reproduction, metabolism, oncogenesis and immunity, the nomenclature and taxonomy of Fox proteins are reviewed. In addition, the molecular structure of Fox proteins and chemical modification and regulation of FOXO subfamily is summarized. Furthermore, the functions of FOXO proteins in apoptosis and oncogenesis are discussed.

Abstract:Palladin is a novel actin-associated protein, its distribution is ubiquitous in smooth muscle ， central nervous system and embryonic tissues. It seems that palladin is required for the establishing/maintaining actin cytoskeleton and its dynamic assembly. Palladin colocalizes with alpha-actinin in actin cytoskeleton. Findings show that palladin play a critical role in controlling cell shape, cell migration or movement. Palladin expression is upregulated in metastatic cancer cells and astrocytes following traumatic injury to the central nervous system. Palladin upregulation allows the astrocytes to form the glial scar.

Abstract:To study the prevention of dauricine (Dau) on bradykinin (BK) induced alteration of intracellular calcium homeostasis and tau phosphorylation, fluorescence spectrophotometer with dual excitation was utilized to measure the intracellular calcium concentration ([Ca2+]i), MTT to detect cell viability and immuncytochemistry to examine tau phosphorylation. The results showed (1) cells treated with BK 1 μmol/L induced a transit increase in [Ca2+]i in all the cell lines detected, among them, the sustained increase of [Ca2+]i level was only seen in PS1 Δ9/APPswe cell at 2 h and 24 h after the treatment. Dau (3 μmol/L or 6 μmol/L) prevented BK-induced transit and sustained elevation and fluctuation of [Ca2+]i; (2) BK treatment decreased the cell metabolism detected at 2 h in PS1Δ9/APPswe and Dau antagonized the effect; (3) BK induces Alzheimer-like tau hyperphosphorylation at tau-1 epitope and Dau partially antagonized this effect. In conclusion, Dau inhibits BK-induced disturbance in intracellular calcium homeostasis and tau hyperphosphorylation at tau-1 sites.

Abstract:NGX6 is a candidate tumor suppressor gene which was isolated by location candidate cloning strategy. In order to explore the effect of NGX6, the mammal expression vector of NGX6/pcDNA3.1(+) was transfected into 5-8F cell （ with high ability of metastasis ） by liposome. The integration of the exogenous vector DNA and the expression of NGX6 were detected by Northern blot and RT-PCR respectively. The cytobiological characterization of positive clone was analyzed by growth curves of cells and soft agar assay. cDNA array techniques were used to profile the potential targets of NGX6. The results showed that 5-8F cells with overexpression of NGX6 grew slower than that of control, and NGX6 could influence the expression of cell cycle, cell adhesion and angiopoiesis molecules, such as up-regulating p19, catenin α2, desmoglein 1,down-regulating EphB4, TIE2, vitronectin. These data revealed that NGX6 might play a role in tumor metastasis in nasopharyngeal carcinoma (NPC).

Abstract:The fused gene (IFN-TFN) of TFN (transferring N-terminal half-molecule) gene and IFN (interferon) gene was amplified by multiple PCR.The fused gene and TFN gene was inserted into pPIC9 vector. The recombinant plasmid pPIC9-IFN-TFN and pPIC9-TFN were transformed into Pichia pastoris GS115 by PEG. After being induced by methanol, the target proteins were expressed in ferment supernatant at high level. The recombinant fused protein IFN-TFN and recombinant TFN with purity respectively being higher than 93% and 95% were finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. According to in vitro bioactivity assay, the fused protein IFN-TFN had antiviral activity but which was much lower than the natural IFN. Fe3+ saturation study confirmed that the recombinant IFN-TFN was able to bind Fe3+ as the recombinant TFN did. It was shown that TFN could be used as the transcellar carrier of IFN.

Abstract:Monooxygenase domain of cytochrome P450BM-3 from Bacillus megaterium was evolved by error-prone PCR. Three mutants (D168N, A225V, K440N; E435D; I39V) with higher hydroxylating activity than the parent type P450BM-3((A74G, F87V, L188Q)) were obtained, coupled with a sensitive screening method of absorption of hydroxylating indole to indigo at 630nm. The catalytic activities of three mutants were 6.6(hml001), 9.6(hml002), 5.3 (hml003) fold higher than that of the parent type P450BM-3 respectively. The kinetic analysis revealed that the mutant enzymes exhibit a higher substrate binding ability and catalytic efficiency than the parent enzyme. DNA sequence indicated that hml001 and hml003 cover one amino acid substitution (I39V and E435D, respectively), hml002 contains three amino acid substitutions (D168N, A225V, K440N).

Abstract:Chemokine receptor-5 (CCR5) serves as a co-receptor necessary for the binding of HIV-1 to the host cells, the defective CCR5 function and the blocking of CCR5 sites by CCR5 antagonists will suppress the entry of HIV-1 to target cells. To acquire the peptide antagonists specifically binding CCR5, CHO cells stably expressing human CCR5 (CHO/CCR5) were used to select CCR5-binding peptides from a phage displayed 12-mer peptide library. After four rounds of selection, eleven out of the 20-phage clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages could competitively bind to CHO/CCR5 cells with anti-human CCR5 mAb, and the synthetic peptide AFDWTFVPSLIL could inhibit RANTES binding to CHO/CCR5. These results suggest that the peptide could specifically bind CCR5 molecules.

Abstract:The Gal: 3-O-sulfotransferase-2 (GP3ST) was a newly cloned sulfotransferase and its biological significance remained unknown. The expression levels in different metastatic potential tumor cells and larynx cancer tissues were investigated. A significantly higher expression level of GP3ST and sulfated glycocojugates were observed in highly metastastic cancer cells and larynx cancer tissues with lymph node metastasis than those in lowly metastatic cancer cells and ones without metastasis (P < 0.05, n = 42). GP3ST expression was further suppressed by targeted RNAi technology in hepatocarcinoma cells SMMC7721. It was found that the morphology of SMCC7721 cells transfected with RNAi-GP3ST was changed from polygon to shuttle shape and the adhesion ability to HUVEC induced by TNF-α and sL-selectin was significantly decreased. Furthermore, the decreased expression of GP3ST could lead to expression inhibition of integrin subunit αV, but didn't affect the expression of integrin subunit β3 by Western blot analysis and RT-PCR. The result of higher αV expression in high metastatic cells than in low metastatic cells was consistant with the differential expression of GP3ST. Taken together the results suggested that the expression of GP3ST may be involved in tumor metastasis process by regulation of adhesion ability and expression of integrin αV.

Abstract:A novel radish RsPHGPx cDNA, which encodes a functional phospholipid hydroperoxide glutathione peroxidase (PHGPx) protein, was identified in the previous work. In the study genomic organization and the upstream regulatory sequence analysis of this gene was presented. Southern blot analysis showed that RsPHGPx gene existed in radish genome in manner of single copy. Moreover, a 3.3 kb genomic DNA fragment of RsPHGPx gene was isolated by combination of common PCR and genome-walking method. Sequence analysis on this genomic fragment demonstrated that RsPHGPx gene consists of seven exons separated by six introns, and suggested that a short 5'-flanking sequence immediately before the exon 1 should be the putative RsPHGPx promoter region, which is proceeded by the upstream neighboring biotin synthase gene. Cis-acting elements search showed that the putative promoter contains elements responsive to hormones (eg. E-Box and W-Box), abiotic stresses (eg. MYB and MYC binding sites), and light (Box Ⅱ and I-Box), etc. Northern blot analysis indicated that the expression of RsPHGPx was subjected to up-regulation of chilling and down-regulation of ABA and successive illumination (in etiolated seedlings), implying the regulatory roles of some predicted elements. However the up-regulation effect of herbicide paraquat, which can induce oxidative stress, suggested the presence of some unknown elements in the promoter region. This is the first report on gene structure and upstream regulatory sequence analysis in reported plant PHGPx genes, which will be a prerequisite to understand regulatory mechanism of PHGPx gene expression in plants.

Abstract:The gene fragment encoding ATR(CMG2)-EXCELL (excellular potion of anthrax toxin receptor/capillary morphogenesis factor) was cloned into a secretory expression plasmid and then expressed in media supernant of Pichia pastoris. The recombinant rATR(CMG2)-EXCELL expressed was about 20% of the total proteins in media supernant. About 1 mg electrophoresis purity rATR(CMG2)-EXCELL could be obtained after the purification of 1 L culure using chelating column. In vitro binding activity analysis and cell protection experiments have shown that rATR(CMG2)-EXCELL has an excellent biological activity. The successful expression of rATR(CMG2)-EXCELL has placed a solid foundation for the research on binding mechanism of ATR and PA(protective antigen) and developing new cure for anthrax.

Abstract:In order to provide a rapid, selectivity and very high sensitivity method for the determination of ochratoxin A (OTA), an indirect competitive time-resolved fluoroimmunoassay(TRFIA) was used. Tests were performed in a 96-well microplate using the self-produced toxin-specific monoclonal antibodies 3G9, obtained from mice immunized with ochratoxin A - bovine serum albumin (OTA-BSA). In indirect TRFIA format, OTA-BSA was coated onto the microtitre plate and incubated with standard toxin and anti-OTA antibody. A goat antimice IgG- Eu3+ conjugate was used to enable detection. The suitability of the assay for quantification of OTA was also studied. Results showed ascitic fluids could be used at a dilution exceeding 1:10 000 and the OTA detection limit to be 0.03 μg /L for indirect competitive TRFIA formats. The 80%, 50% and 20% inhibition binding ffective dose(ED80、 ED50、 ED20) of OTA were (0.33±0.02) μg/L, (1.44±0.08) μg/L and 5.22±0.12) μg/L. The assay range was 0.03 ~ 1 000 μg /L . The cross reactivity with ochratoxin B was 3.7% and the antibodies did not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OTA- TRFIA were 3.7% and 5.3% respectively. The mean recoveries from OTA-free cereals spiked with 1 ~ 200 μg/kg OTA of cereals sample were 94.2%. Both OTA- TRFIA and OTA-ELISA test were applied for the quantitative measurement of OTA in the same cereals, and the coefficient of correlation was 0.925. It was shown that the newly developed TRFIA could be applied to detect the OTA contamination in cereals. The OTA-TRFIA provides very high sensitivity and optimal range, and it will be useful to screen OTA contamination easily, simply and economically when the number of samples is large.

Abstract:MALDI-TOF is a simple and robust method for the analysis of single nucleotide polymorphism (SNP), which combines proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. This technology is among the most promising SNP typing methods for delivering accurate, effective, flexible and high throughput analysis of SNPs. “4-plex” genotyping protocol, which could detect 4 SNPs in one reaction, was performed in Beijing Genomics Institute with accuracy of 99.55%. To learn the correlation between genotyping call rates and quality, nearly ten thousands of typing results (“4-plex”) were analyzed. Significant positive correlation between call rate and the counts of conservative results were observed. SNPs with call rate lower than 82% have less conservative results, suggesting 82% was a cutoff to evaluate the genotyping results of MALDI-TOF assay. Aiming to further improve genotyping throughput and reduce the cost, “Mix 8-plex” and “Double-spotting 8-plex” genotyping protocols were developed. For new protocols, PCR and primer extension reactions were still performed under reliable “4-plex” protocol. For “Mix 8-plex” protocol, two sets of “4-plex” primer extension products were mixed and dispensed onto one SpectroCHIP at the same time. As to “Double-spotting 8-plex” protocol, two sets of “4-plex” products were consecutively dispensed onto one SpectroCHIP. 32 SNPs were genotyped in 95 human DNA samples to test the feasibility of these new protocols. The results showed that the performance of “Mix 8-plex” was as good as “4-plex” protocol, while the performance of “Double-spotting 8-plex” was poor.