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P Tien

Publications and source records attributed to P Tien.

At least 19 recordsLinked to original sources
PubMed2003

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus.

Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials.

Amino Acid Sequence↗
PubMed2001

Tomato yellow leaf curl China virus: monopartite genome organization and agroinfection of plants.

The complete DNA sequence (2734 nucleotides) of the monopartite genome of tomato yellow leaf curl China virus (TYLCCNV), a begomovirus transmitted by the whitefly Bemisia tabaci, was determined. The circular genomic DNA contains six open reading frames (ORFs) encoding proteins of molecular weights >10 kDa, of which two (V1 and V2) are located on the virion-sense strand and four (C1, C2, C3 and C4) on the complementary-sense strand. The ORFs are comparable to those of other whitefly-transmitted begomoviruses with a monopartite genome and to those encoded by DNA-A of bipartite begomoviruses. Sequence comparisons with other geminiviruses showed that TYLCCNV belongs to Begomovirus from the Old World. No putative DNA-B genome was found. Nicotiana species and tomato plants agroinoculated with the TYLCCNV monopartite genome developed typical yellowing and leaf-curling symptoms. The cloned molecule carried all the information needed for virus replication and systemic infection of plants.

China↗
PubMed2001

Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme.

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.

Arachidonate 12-Lipoxygenase↗
PubMed2001

HBV-specific peptide associated with heat-shock protein gp96.

Glycoprotein 96 (gp96), a member of the heat-shock protein family, can elicit priming of antigen-specfic cytotoxic T lymphocytes, when bound to antigenic viral or tumour peptides. We used direct peptide isolation from purified gp96 and microsequencing to show that a virus-specific peptide is bound to gp96 derived from liver tissues of patients with hepatitis B virus (HBV)-induced hepatocellular carcinoma. This virus-specific peptide has potential for engineering tumour vaccines against hepatocellular carcinoma and chronic HBV infection.

Antigens, Neoplasm↗
PubMed2001

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus.

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.

Amino Acid Sequence↗
PubMed2001

[Cloning and sequencing of Chinese squash leaf curl virus].

Sequence analysis of virus isolation DNA of Squash leaf curl disease shows that Squash Leaf Curl Virus (SLCH-CHI) contains 2741nt, which encode six Open Reading Frames (ORFs). Two of them are on virus sense strand: AV1 (256aa) and AV2 (140aa). AV1 encode coat protein. The other of ORFs are on complementary sense strand: AC1 (replicase gene, 243aa), AC2 (transactivtor, 134aa), AC3 (136aa) and AC4 (172aa). The virus belongs to one number of begomoviruses from old world.

Amino Acid Sequence↗
PubMed2000

Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication.

Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China. Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity. A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III. By biopanning, the fused peptides were bound to the biotinylated GCHV. Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91. Three rounds of affinity selection enriched the pool of inhibiting peptides. Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected. The TCID(50) of GCHV was decreased over 10000x. Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH(2)-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH. A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro.

Animals↗
PubMed2000

Ribozyme-mediated resistance to rice dwarf virus and the transgene silencing in the progeny of transgenic rice plants.

A hammerhead ribozyme (Rz) with long hybridizing arms targeting the mRNA of rice dwarf virus (RDV) segment 5 and a mutated nonfunctional ribozyme (mRz) were constructed. As predicted, Rz transcribed in vitro cleaved the target mRNA of RDV segment 5 into two fragments of 138 and 238 nucleotides in length. The Rz and mRz genes were each placed under the control of the CaMV 35S promoter and used to transform Japonica rice variety 'Tongling No. 1' via Agrobacterium tumefaciens. A total of 32 independent lines containing Rz or mRz was obtained as demonstrated by Southern blot analysis. Challenge inoculation with RDV viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants containing the Rz transgene displayed high resistance or delayed and attenuated viral symptoms. In contrast, transgenic lines expressing mRz showed severe symptoms similar to the control plants transformed with the vector alone. These results suggest that Rz confers RDV resistance in transgenic rice. Genomic DNA PCR analysis confirmed that all of the examined T6 progeny plants contained the Rz transgene. However, accumulation of the Rz transcripts was detectable by RT-PCR only in the plants that were resistant to RDV. This suggested that loss of RDV resistance in progeny plants containing the Rz transgene may result from silencing of the Rz transgene.

Base Sequence↗
PubMed2000

Ribozyme-mediated suppression of platelet type 12 lipoxygenase in human erythroleukemia cells.

The platelet type 12 lipoxygenase (12-LOX) adds molecular oxygen to C-12 arachidonic acid to yield 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. It has been suggested that 12-LOX and its metabolites play an important role in tumor angiogenesis. A hammerhead ribozyme (Rz) targeted to the first GUC site within the 12-LOX mRNA was designed and cloned into an in vitro transcriptional or mammalian expression vector. In vitro, the Rz was able to cleave its substrate efficiently in a time-dependent manner. Under multiple turnover conditions, the Rz performed well at 37 degrees C, with a further improvement at 50 degrees C. When cloned into a mammalian expression vector, pSV2neo, the Rz construct efficiently decreased the level of 12-LOX mRNA in stably transfected human erythroleukemia cells to levels that were undetectable by Northern blot analyses. 12-LOX enzyme activity assays showed that Rz significantly reduced the 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid production in human erythroleukemia cells; this effect was sustained for up to 6 months in cell culture. The Rz developed in this study may represent a powerful tool for potential applications, ranging from an understanding of tumor angiogenesis to cancer gene therapy.

Arachidonate 12-Lipoxygenase↗
PubMed2000

[Ribozyme which specifically cleave 12-lipoxygenase mRNA: in vitro activity and its dynamics].

The ribozyme gene which specifically cleaved 12-lipoxygenase mRNA was designed, synthesized and cloned according to hammer-head ribozyme model. A 25-bp-long segment of 12-lipoxygenase mRNA was chemically synthesized as substrate, which was incubated with transcribed ribozyme RNA to detect its cleavage activity in vitro. The results showed that the ribozyme had a relatively high cleavage activity against 12-lipoxygenase mRNA segment at 37 degrees C. It's kcat value and Km value was 0.083/min and 1300 nmol/L respectively. The ribozyme had a very high activity at 50 degrees C and its kcat value was 0.31/min.

Arachidonate 12-Lipoxygenase↗
PubMed2000

[Role of heat shock protein-peptide complexes on tumor and infectious diseases immunity].

Many heat shock proteins, e.g. gp96, HSP90, HSP70, etc have elicited rejection and immunotherapy immunogenicity of tumor and infectious diseases. Further study indicated that hsps can chaperone the endogenous repertoire of peptides, and the antigenicity of hsp-peptide complexes lies in the peptides, not HSPs. HSPs present peptides associated with them to MHC class I molecules for recognition by CTL and memory T cells, and elicit cellular immune responses. The latest finding shows that gp96 may present antigenic peptides directly to T lymphocytes functionally as MHC. In recent years the mechanism of immunogenicity and advantages as vaccine therapy of gp96 and HSP70, the two main hsps in mammals have been studied in detail, which offers a new opportunity for immunotherapy of hepatitis B and hepatocellular carcinoma.

Antigen Presentation↗
PubMed2000

[The regulation activity of Chlorella virus gene 5' upstream sequence in Escherichia coli and eucaryotic alage].

The 5' upstream regions of adenine methyltransgerase gene and major coat protein gene (PAMT, PVP54) in Chlorella virus genomes were used to contract transformation vectors in E. coli and eukaryotic algae. The regulation activities of PAMT and PVP54 comparing with PRPL and CaMV35S promoters were analyzed in different E. coli strains and Chlorella species. It is found that the luciferase activity controlled by PAMT is 50-400 times higher than that controlled by PRPL. The regulation activity of PAMT in 2 Chlorella species is obviously higher than that of CaMV35S promoter. It is the first report that the 5' upstream region of Chlorella virus gene has strong regulation activity in eucaryotic algae. The result suggests this regulation sequence will have an excellent application in the eucaryotic algae genetic engineering.

Chlorella↗
PubMed2000

[cDNA cloning, fusion expression in Escherichia coli and activity assay of hIL-11].

Human Interleukin-11 (hIL-11) is a multifunctional cytokine which plays an important role in regulating the proliferation and differentiation of cells in the hematopoeitic, lymphoid system etc. To obtain the IL-11 cDNA, a primary culture of Chinese fetal lung fibroblast was prepared from fresh tissue. Then the human IL-11 cDNA without the N-terminal signal peptide sequence was cloned by RT-PCR from the cells induced by PMA. The sequence indicated that there are three bases different from those previously reported, but with no change of the amino acids. The cDNA was inserted into the 3' end of trxA gene in thioredoxin gene fusion expression system pTRXFUS to construct the trxA and hIL-11 fusion expression vector, and expressed in E. coli. The fusion hIL-11 accounts for more than 20% of the total bacteria proteins. The expression product is present in soluble forms and has the full biological and immunological activities.

Cells, Cultured↗
PubMed1997

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes.

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50 degrees C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Base Composition↗
PubMed1996

Infection and replication of a planthopper transmitted virus-rice stripe virus in rice protoplasts.

Rice stripe virus (RSV), a planthopper-transmitted virus, was inoculated into rice protoplasts, and a one-step growth curve was determined. The amount of virus in the protoplasts decreased following the inoculation, and then increased after 8 h. The replication of RSV reached its peak 20 h after inoculation. RSV replication in the inoculated protoplasts was further demonstrated by electron microscopy.

Animals↗
PubMed1996

Immunodetection of beet necrotic yellow vein virus RNA3-encoded protein in different host plants and tissues.

The protein p25 open reading frame (ORF) of beet necrotic yellow vein virus-BNYVV RNA3 was cloned into bacterial expression vector downstream of the 5-'terminus part of beta-galactosidase ORF and the expressed p25 fusion protein was used to produce an antiserum. The latter was employed to detect the subcellular location of p25 in mechanically inoculated Tetragonia expansa, Chenopodium quinoa and sugarbeet leaves by Western blot assay. The results showed that p25 was present as a soluble protein only in the S30 fraction of T. expansa, C. quinoa and sugarbeet leaves infected with BNYVV.

Animals↗