Biomedical subjects

Yi Fu

Publications and source records attributed to Yi Fu.

23 records · Page 2Linked to original sources

PubMed2003

Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction.

Human parvovirus B19 (B19) infects cells of erythroid lineage. Production of neutralizing antibodies (Abs) is indispensable for recovery from B19-related disease state. In this study, we used a convenient method to measure neutralizing activities in human sera by using a real-time quantitative PCR based assay. Erythroid cell line KU812Ep6 was incubated with test sera before infection with B19 virus. The copy number of B19-DNA in cultures was decreased in the presence of the sera from patients who recovered from acute B19 infection, whereas no decrease in B19-DNA was in cultures incubated with sera from healthy volunteers who had no B19 infection. The decrease in B19-DNA copy number was calculated and the inhibition percentage was expressed as neutralizing activity to B19. A clinical study showed that the levels of neutralizing ability were high in patients who recovered soon after acute B19 infection, but were low in some patients with a prolonged clinical course for recovery from B19 infection. This method is simple and convenient compared with methods described previously, showing its usefulness to evaluate the neutralizing activity to B19.

Animals↗

PubMed2003

Identification of important amino acid residues for human IL-18 function by mutant construction.

To study the structure-function relationship of IL-18, two IL-18 mutants, N- and C-terminal mutant (Delta NC) and IL-1 signature-like sequence mutant S(154)A/Y(156)F/E(157)P/C(163)T (S), were constructed by PCR. The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, purified by Sephadex G-75 chromatography and renatured by stepwise dilution. The purity of the recombinant proteins was over 95%. The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma (IFN-gamma) production and NF-kappa B activation from human peripheral blood mononuclear cells (PBMC). Our results showed that the two mutants induced significantly less amount of IFN-gamma from PBMC (13%, 48% of wild type rhIL-18 for Delta NC, S, respectively), and the activation of NF-kappa B also lower than wild type rhIL-18(69.7%, 89.8% of wild type rhIL-18 respectively), indicating that the deleted or mutated amino acids might be important for IL-18 function.

Amino Acid Sequence↗

PubMed2002

Regulation of tumor necrosis factor alpha promoter by human parvovirus B19 NS1 through activation of AP-1 and AP-2.

Human parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-alpha), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-alpha, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-alpha mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-alpha promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-alpha transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-alpha expression would explain the pathogenesis of B19-associated inflammation.

DNA-Binding Proteins↗

PubMed2002

[Site-directed mutagenesis of the cysteines of human IL-18 and its effect on IL-18 activity].

To study the structure-function relationships of human IL-18(hIL-18), site-directed mutagenesis was used to generate four hIL-18 cysteine mutants, C74S, C104S, C112S and C163S. The cDNAs of the four cysteine mutants were inserted into prokaryotic expression vector pJW2 and expressed as inclusion bodies in E. coli. The inclusion bodies were washed with 2 mol/L urea, dissolved in 8 mol/L urea, and purified by chromatography on Sephadex G-100 column. The purity of the purified mutants were greater than 90% as judged by SDS-PAGE. The activity of rhIL-18 C74S, C104S, C112S and C163S accounted for 5%, 81%, 58% and 11% of wild type, respectively. These results suggest that Cys74 and Cys163 play important roles in inducing IFN-gamma production in human peripheral blood mononuclear cells.

Amino Acid Substitution↗

PubMed2001

Asp(126), Asp(130) and Asp(134) are Necessary for Human IL-18 to Elicit IFN-gamma Production from PBMC.

To identify the amino acid residues which are critical to interleukin 18 (IL-18) function, three highly-conserved amino acids (Asp(126), Asp(130) and Asp(134)) were mutated to Asn, Lys and Lys. The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, renatured by stepwise dilution and purified by Sephadex G-75 chromatography. The purity of the recombinant proteins was over 95% and Western blot showed that the mutant rhIL-18 had the same immunogenicity as that of wild type rhIL-18. The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma(IFN-gamma) production from human peripheral blood mononuclear cells(PBMC). The results showed that the three mutants induced significantly less amount of IFN-gamma from PBMC(32%, 8% and 10% of wild type for hIL-18D(126)N, hIL-18D(130)K and hIL-18Df(134)K, respectively) indicating that the three highly conserved amino acids are necessary for human IL-18 function.

Journal Article↗