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Biomedical subjects

P Tian

Publications and source records attributed to P Tian.

At least 19 recordsLinked to original sources
PubMed2006

Detection of norovirus capsid proteins in faecal and food samples by a real time immuno-PCR method.

AIMS: To develop a sensitive real time immuno-polymerase chain reaction (rtI-PCR) method for detecting norovirus (NV) capsid protein in food samples. METHODS AND RESULTS: The viral antigens were captured by two polyclonal antisera against recombinant Norwalk viral-like particles (rNVLPs). Biotin-conjugated antibodies, avidin and biotin-conjugated DNA reporter were used to convert the protein signals into DNA signals. The reporter DNA was then amplified by addition of primers and PCR. A real time PCR method was used in order to perform a quantitative post-PCR analysis. One hundred rNVLPs (10 fg) and a NV sample containing 660 rNVLPs equivalent particle units (66 fg) could be detected by this method. CONCLUSION: The PCR inhibitors present in the food samples had minimal effect on antigen capture and were removed by multiple wash steps during the rtI-PCR procedure. The sensitivity of rtI-PCR was >1000-fold higher than the standard enzyme-linked immunosorbent assay and approximately 10 times higher than reverse transcription PCR in detection of NV capsid protein in stool and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a rtI-PCR method to detect NV in contaminated food samples without concentration or purification of the virus.

Animals↗
PubMed2006

Norovirus binds to blood group A-like antigens in oyster gastrointestinal cells.

AIMS: To determine if histo-blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells. METHODS AND RESULTS: HBGA-specific monoclonal antibodies (MAbs) were used to determine the presence of the corresponding HBGA in oyster GI cells. All oyster samples tested contained type A-like HBGA in GI tissue as measured by ELISA. Recombinant Norwalk virus viral like particles (rNVLP) were bound to plates coated with oyster GI homogenate. The binding was inhibited when rNVLPs were pre-incubated with MAbs specific for type A HBGA, or samples of human saliva from type A individuals. Co-localization of rNVLP and type A-like HBGA, but not type B-like or type H-like HBGA, on GI epithelial cells was observed by immunofluorescent histochemical staining and three-channel confocal scanning laser microscopy. CONCLUSION: Type A-like HBGA is present in oyster GI cells and responsible for binding of rNVLP. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the presence of type A-like HBGA in oyster GI cells and the specific binding of rNVLP to type A-like HBGA on oyster GI cells. The results of this study suggest that human NoV concentrate in oyster GI cells by specific binding to concentrated type A-like HBGA rather than by a nonmolecular entrapment within the tissues.

ABO Blood-Group System↗
PubMed2005

Porcine gastric mucin binds to recombinant norovirus particles and competitively inhibits their binding to histo-blood group antigens and Caco-2 cells.

AIMS: To determine if human noroviruses (NV) bind to histo-blood group antigens (HBGA) from pig gastric mucosa. METHODS AND RESULTS: An assay was developed to measure the inhibition of binding of recombinant norovirus-like particles (rNVLP) to HBGA in human saliva by porcine gastric mucin (PGM). The binding of rNVLP to HBGA could be inhibited by PGM in a dose-dependent pattern. Also, rNVLP could be captured effectively by PGM coated directly on plates and was detected by binding of polyclonal antibodies against rNVLP. Similarly, the binding of rNVLP to PGM could be inhibited effectively by HBGA in human saliva, and by Lewis b and Lewis d synthetic oligosaccharides (OS), but not inhibited effectively by an H3 OS or by purified bovine submaxillary gland mucin. Preincubation of rNVLPs with PGM completely inhibited their binding to human Caco-2 cells. CONCLUSIONS: PGM binds effectively to rNVLPs and competitively inhibits rNVLPs binding to human HBGA and Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the binding of glycoproteins from animal gastric mucosa to human NVs. This study highlights the importance of further characterizing the NV incidence and infections in nonhuman animal hosts and the possibility that NV is a zoonotic disease.

Animals↗
PubMed2001

[Immunorelated pancytopenia].

OBJECTIVE: A report of a group of patients with pancytopenia which might be related to abnormal immunity. METHODS: The clinical and laboratory features of 29 patients with positive bone marrow mononuclear cell (BMMNC) Coombs tests were analyzed. RESULTS: Most of these cases were pancytopenia with normal or decreased bone marrow cellularities and increased normoblasts. They were all found to have negative results of conventional hemolysis tests and hematopoietic nutrient determination and had no evidence of malignant clonal hematopoiesis. However, all of them showed positive BMMNC-Coombs test and good response to corticosteroids. CONCLUSION: Immunorelated pancytopenia might be caused by abnormal immunity (especially autoantibody) mediated destruction or abnormal function of blood cells. It is beneficial to differentiate immunorelated pancytopenia from aplastic anemia or myelodysplastic syndromes.

Adolescent↗
PubMed2001

[Expression of two truncated enhancin gene from Helicoverpa armigera granulosis virus in E. coli and its preliminary bioassay].

The plasmids pET-30a-Ben and pET-30a-Den which included 1.7 kd and 2.2 kb fragments of 5'-terminal of HaGV enhancin gene were obtained by cutting recombinant plasmid pET-30a-En with Bal I and Dra I respectively. Two fragments were expressed in E. coli successfully and the products were named Ben and Den respectively. The enhancement, which Ben and Den enhance the infectivity of HaNPV and Bt in 3rd larvae of Helicoverpa armigera, was studied. The results indicated that there was increase of the mortality of 10.5%-26.5% and the LT50 decrease of 0.9 d causes by adding Ben, while Den could increase the mortality by 10.2%-33.0% and decrease the LT50 by 1.2 d-1.9 d. The preliminary bioassay on Bt against Helicoverpa armigera indicated the recombinant enhancin could increase the mortality of larvae by 20.7%-35.4%, Den by 16.7%-31.5%, Ben by 11.7%-27.4%.

Animals↗
PubMed2001

[Construction of mucosal vaccine derived from HBV surface antigen epitope A and the assay of its immunogenicity].

The fusion gene of HBV(adr) surface antigen epitope and B subunit of Cholera toxin was constructed and expressed successfully in E. coli at high yield. After denaturation-renaturation process, SDS-PAGE analysis showed that most of the renatured product reassociated in a pentameric form which was the same as natural CTB. Western blot analysis indicated that the immunogenicity of HBsAg antigen epitope was conserved. Moreover, ELISA analysis of the sera of orally, intranasally and intraperitoneally immunized mice showed that the circulating IgG antibodies to HBsAg were developed. The results may be helpful for constructing the novel mucosal vaccine with high efficacy.

Animals↗
PubMed2000

Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site.

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.

Amino Acid Sequence↗
PubMed2000

The authentic sequence of rotavirus SA11 nonstructural protein NSP4.

Recent studies demonstrate that the rotavirus nonstructural protein NSP4 functions as an enterotoxin and plays an important role in viral pathogenesis. Previous in vitro studies of NSP4 have used a cDNA clone of gene 10 derived from the prototypic rotavirus strain, SA11. We recently compared the sequence of the commonly used NSP4 cDNA with the sequence obtained from several SA11 isolates by direct sequencing of reverse transcription polymerase chain reaction products. One codon difference was identified between the cDNA clone and the SA11 virus isolates, and this resulted in a predicted amino acid substitution at position 47. The cDNA sequence specifies an asparagine at position 47, and the SA11 virus gene 10 encodes a hisitidine. To determine if this amino acid substitution altered the function of NSP4, we analyzed the ability of both NSP4-Asn47 and NSP4-His47 to regulate intracellular calcium levels and exhibit cell cytotoxicity. Our results indicate that the expression of NSP4-His47 from a recombinant baculovirus displays enhanced cytotoxicity and calcium flux.

Amino Acid Substitution↗
PubMed2000

Sperm function tests after vasovasostomy.

AIM: To evaluate the sperm function after vasovasostomy. METHODS: Semen samples from 42 subjects after vasovasostomy (Group A: 1-6 months, Group B: 6-12 months; Group C: 12-18 months after vasectomy reversal) were investigated. Semen from 34 normal fertile men was used as controls. Sperm function tests, including hypoosmotic swelling test (HOST), acridine orange (AO) fluorescence, acrosome reaction (triple-stain), cervical mucus penetration test (CMPT), etc were done. RESULTS: After vasectomy reversal, the percentage of HOST was significantly lower than that of the normal fertile men. In regard to AO, there were no significant differences between the three vasovasostomy groups and between these 3 groups and the controls. With triple-stain, the percentage of normal acrosome reaction was significantly lower in Group A as compared with the controls, but not in Groups B and C. There were no significant differences in the results of CMPT between the vasovasostomy groups and the controls. However, the number of "poor" type was significantly higher in Groups A and C than in the controls; the percentage of "negative" type were higher in Groups A and B than in the controls. CONCLUSION: After vasovasostomy a lower level of HOST remained for one year and gradually recovered after one year. Six months after vasectomy reversal, the percentage of acrosome reaction could be changed from lower level to normal range. The data of AO indicated that the genetic material (double-stranded DNA) in spermatozoa was not affected by vasovasostomy. To evaluate the result of CMPT after vasectomy reversal, not only the normal results but also the abnormal results ("poor" and "negative" types) should also be considered.

Adult↗
PubMed2000

[Modified vascular endothelia growth factor receptor-mediated targeting nonviral gene delivery system and in vivo beta-gal transducing experiment].

OBJECTIVE: To investigate whether the modified vascular endothelial growth factor (VEGF) receptor-mediated targeting nonviral gene delivery system is efficient in transducing exogenous gene. METHODS: We detected the expression of VEGF receptor on some tumor models by immunohistochemistry at first and selected nine models, i.e. SMMC-7721, SK-OV-3, H128, SPC-A1, LoVo, MKN-45, BCaP-37, A375, cervical cancer xenograft and hepatoma xenograft. Then we prepared two complexes, GV1-P.L./beta-gal and GV2-P.L./beta-gal, and injected the two complexes subcutaneously into nude mice around the tumor. The expression of the reporter gene was observed in the vascular endothelial cells and some tumor cells with highly expressed VEGF receptor. RESULTS: GV2 gene delivery system could transduce exogenous gene into vascular endothelial cells and subcutaneously transplanted tumors such as SK-OV-3, and cervical xenograft which were rich of VEGF receptor on the surface of the tumor cell. The expression reached a high level at day 3 and decreased eventually until the 3(rd) week. There was no expression in the heart and lung but low expression was seen in the spleen. GV1 gene delivery system also transduced exogenous gene into the endothelial cells but with low efficiency. CONCLUSION: This system can specifically transduce beta-gal to cells rich of VEGF receptor but not to cells with undetectable VEGF receptor. The targetability of this modified system is increased as compared to the system previously reported.

Animals↗
PubMed2000

[Molecular cloning of enhancin gene from Helicoverpa armigera granulosis virus and its expression in E. coli].

In order to provide recombinant enhancin for studying its mechanism of increasing the mortality of larvae infected by HaNPV and creating new insecticide, enhancin gene from Helicoverpa armigera granulosis virus was amplified by PCR technique. 2.7 kb fragment of enhancin gene was cloned into EcoRI/XbaI site of plasmid pBluescript KS. Sequence analysis revealed that enhancin gene was similar with that reported in the literature except eight nucleotides and five amino acids. Thereby enhancin gene was inserted into vector pET-30a and expressed successfully in Escherichia coli BL21(DE3). The preliminary bioassay of expressed product and HaNPV indicated that mortality of larvae increased 31.7%-34.1% in 7 post-infection days and the LT50 decreased at least 1.5-2.1 days.

Amino Acid Sequence↗
PubMed1998

[HIM82 protects RBC from the ROS damage produced by activated PMN].

OBJECTIVE: To investigate the protection of RBC from ROS damage produced by activated PMN with McAb-HIM82. METHODS: RBC was injured by ROS produced from PMN activated with PMA and the injury was antagonised by adding HIM82(30 micrograms/ml) to the medium with PMN. RESULTS: Comparing antagonitic group with injured group, the activity of AchE on RBC membrane after adding HIM82 was recovered from (71.36 +/- 22.15)% to (87.66 +/- 14.78)% (n = 6 P < 0.001). The proportion of MDA was decreased from (52.42 +/- 20.63)% to (23.81 +/- 12.4)%(n = 9 P < 0.001); the hemolysis rate was decreased from (80.76 +/- 20.23)% to (66.30 +/- 7.08)% (n = 6 P < 0.05); CONCLUSIONS: The result showed that the McAb HIM82 could protect RBC from injury by down-reguration of ROS level in activated PMN. It could help to establish a newer approach to study the regulation effect of McAb HIM82 on ROS production from PMN actived with relerant cytokines as well as mediators.

Acetylcholinesterase↗
PubMed1996

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114-135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Aging↗
PubMed1996

Rotavirus protein expression is important for virus assembly and pathogenesis.

Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.

Animals↗
PubMed1996

The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity.

During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum (ER). As rotavirus particles mature, they lose their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell. The nonstructural glycoprotein NSP4 functions as an intracellular receptor in the ER membrane (K. S. Au, W. K. Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), and it has been hypothesized that NSP4 is involved in the removal of the envelope during viral morphogenesis (M. K. Estes and J. Cohen, Microbiol. Rev. 53:410-449, 1989; B. L. Petrie, M. K. Estes, and D. Y. Graham, J. Virol. 46:270-274, 1983). The purpose of the present study was to determine if NSP4 has a direct membrane destabilization activity (MDA) by using liposome leakage assays and electron microscopic visualization of liposome, microsome, and viral envelope disruption. The fluorescent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4. A region corresponding to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes. NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein. These results suggest that this region contains at least part of the functional domain of NSP4. Liposomes composed of phosphatidylcholine and microsomes (to simulate ER membranes) were broken when observed by electron microscopy after incubation with NSP4 or the NSP4(114-135) peptide. In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and erythrocytes were not disrupted by NSP4 and the NSP4(114-135) peptide. These results provide direct evidence that NSP4 possesses MDA and suggest that it can cause ER membrane damage. Therefore, NSP4 might play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. A model for the MDA of NSP4 in viral morphogenesis is proposed.

Amino Acid Sequence↗
PubMed1995

The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum.

We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗