Biomedical subjects

X X Peng

Publications and source records attributed to X X Peng.

At least 19 recordsLinked to original sources

PubMed2006

Immunocapture UPPCR combined with DGGE for rapid detection of Shigella species.

AIMS: To develop an immunocapture universal primer PCR (iUPPCR) combined with denaturing gradient gel electrophoresis (DGGE) and evaluate it as a method permitting rapid detection of Shigella species and their serotypes. METHODS AND RESULTS: This method amplifying the conserved regions of bacterial 16S rRNA genes of different species or serotypes of Shigella dysentery bacilli captured and enriched by polyvalent antibodies can detect and distinguish causative pathogens rapidly. Four serotypes from three Shigella species including Shigella dysenteriae serotype 1, Shigella boydii serotype 1, Shigella flexneri serotypes 1a and 3a were examined. CONCLUSION: Our approach could be adopted for not only axenic bacterial population but also mixed communities and achieve rapid detection of various bacteria from the same genus or species in one sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The iUPPCR-DGGE method was shown to be more convenient than serotype-specific-antibody-based method of iUPPCR for Shigella species detection and it could be also applied to the quick detection for other kinds of pathogens with many serotypes.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Analysis of hepatitis B virus-immunoglobulin isotype complexes by a novel immuno-capture polymerase chain reaction method.

Hepatitis B virus (HBV) can be present in the circulating blood either as free virus or as a virion-immunoglobulin (Ig) complex. Presently, it remains unclear what specific role each Ig plays in the clearance of HBV. In this study, a novel method that combined immuno-capture and polymerase chain reaction (PCR) amplification was used for detecting and distinguishing different HBV-Ig complexes. Three isotypes of Ig (IgM, IgG and IgA) bound to HBV were detected in the four clinically defined stages of HBV infection in 108 patients. The results showed that all the three isotypes of Ig could bind to HBV, and the patterns of HBV-Ig complexes varied according to disease categories. Interestingly, the frequency of HBV DNA-Ig complexes in hepatitis B e antigen (HBeAg)-positive patients was significantly lower than that in HBeAg-negative patients. All the data suggest that the three isotypes of HBV DNA-Ig circulating immune complex (CIC) may have different biological meanings. In summary, HBV bound to an antibody is a common feature of hepatitis B, and immuno-capture PCR is a valuable method for the analysis of the composition of the immune complexes. The detection of HBV-Ig complexes may provide new and valuable insights into HBV pathogenesis.

Adolescent↗

PubMed2003

Analysis of complement-bound hepatitis B virus complexes by an immuno-capture polymerase chain reaction method.

Hepatitis B Virus (HBV) can be present in the circulating blood either as a free virus or as a virion-immunoglobulin (Ig) complex with or without complement. However, information regarding the complement-bound HBV circulating immune complexes (CIC) in HBV-infected patients is currently not available. In the present study, we have combined immuno-capture and polymerase chain reaction (PCR) and developed a quick method of high specificity for the detection of complement-bound HBV CIC. We found that the frequency of HBV-factor B was associated with clinical types of hepatitis B (HB) but not with that of HBV-C1q. Moreover, increased frequency at P < 0.05 were found for HBV/C1q-CIC in the group with normal total bilirubin (TBIL) and for HBV/factor B-CIC in the group with positive hepatitis B e antigen (HBeAg). These findings suggest that the immuno-capture PCR (iPCR) for the detection of HBV-bound CIC is a valuable method for analysis of the composition of the immune complexes and for the understanding of host immune response and immune pathogenesis in HBV-infected individuals. In summary, iPCR is a valuable method for analysis of the composition of the immune complexes, which may provide new and valuable insights into HBV pathogenesis.

Adolescent↗

PubMed2002

Analysis of HCV-immunoglobulin isotype complexes provide new insights into antibody response to HCV.

Hepatitis C virus (HCV) is known for its ability to establish persistent infection and cause chronic hepatitis in most infected individuals. The antibody response to HCV in HCV-circulating immune complexes (CIC) is unknown. In the present study, we have characterized distinct changes in patterns of HCV-immunoglobulin (Ig) constituents with disease category, viral mutation and clinical markers. The number of samples positive for single HCV-Ig, HCV-IgG and HCV-IgA, HCV-IgM and HCV-IgA, HCV-IgM and HCV-IgG, HCV-IgM, HCV-IgG and HCV-IgA in 47 samples tested were 8 (17%), 1 (2.1%), 9 (19.1%), 4 (8.5%) and 17 (36.2%), respectively. The occurrence of HCV-IgM and HCV-IgA in combination of two isotypes of HCV-Ig became predominant. These results show that defective IgG in HCV-CIC may contribute to long-term viremia. Further analysis indicated that the frequency of HCV RNA/IgA-CIC in the abnormal aspartic aminotransferase (AST) group was significantly higher than that of the normal AST group, and HCV RNA/IgA-CIC frequency in the abnormal alanine aminotransferase (ALT) group was slightly higher than that in the normal ALT group. IgA complexes may reflect the damage degree of liver function during the course of HCV infection. We also found that there were more mutations in supernatant than in other constituents from single-strand conformation polymorphism (SSCP) analysis. Our results suggest that Ig-complexed virions and free virions may have different biological consequences, with the latter being elusive to immunological elimination. The findings in this study may provide some new insights into antibody response to HCV.

Adolescent↗

PubMed2002

Analysis of complement-bound HCV complexes using a novel immuno-capture RT-PCR method.

Recently, more and more evidence has supported the hypothesis that liver cell injury was immune-mediated in patients with hepatitis C virus (HCV) infection, and that circulating immune complexes (CICs) might play a role in the pathogenesis of chronic hepatitis C (HC). In the present study, we have combined immuno-capture and reverse transcriptase-polymerase chain reaction (RT-PCR), and developed a quick method of high specificity for the detection of complement-bound HCV-CIC. We found that there were higher frequencies of HCV-C1q CIC than that of HCV-factor B, and there was a deviation of complement from immunoglobulin (Ig) in HCV-CIC. These findings suggest that immuno-capture RT-PCR (iRT-PCR) for the detection of HCV-bound CIC is a valuable method for the analysis of the composition of the immune complexes, and for the understanding of host immune response and immune pathogenesis in HCV-infected individuals.

Adolescent↗

PubMed2001

Autoimmune responses to mRNA binding proteins p62 and Koc in diverse malignancies.

Two tumor-associated antigens, p62 and Koc, are insulin-like growth factor II (IGF-II) messenger RNA binding proteins. Autoantibodies to p62 have been detected in cancer sera but have not been reported for Koc. This study determined the extent and frequency of autoantibodies to p62 and Koc in diverse malignancies, the epitopes on the antigens, and the presence or absence of cross-reactive antibodies. Recombinant polypeptides were expressed from full-length and partial cDNA constructs and used as antigens in Western blotting, enzyme-linked immunoassay, and immunoprecipitation. After identifying the epitopes, cross-absorption with recombinant polypeptides was used to determine specificity. Sera from 777 patients with 10 different types of malignancy were analyzed. Autoantibodies to p62 were found in 11.6% and to Koc in 12.2% and cumulatively to both antigens in 20.5%, with significant difference from the control populations consisting of normal subjects and autoimmune disease patients (P < 0.01). The immunodominant epitopes were at the amino termini of both antigens and absorption studies showed that the majority of autoantibodies were not cross-reactive. Autoantibodies to p62 and Koc were present in approximately similar frequencies in a variety of malignancies and the immune responses appeared to be independent of each other. The immune responses might be related to overexpression or dysregulation of p62 and Koc in some tumors.

Antigens, Neoplasm↗

PubMed2001

Analysis of HCV-IG isotype complexes by a novel immuno-capture RT-PCR method.

HCV can be present in the circulating blood either as a free virus or as a virion-immunoglobulin (Ig) complex. All isotypes of Igs may form the virus complexes, but it remains unclear what specific role of each Ig plays in the clearance of HCV. In the present study, we have combined immuno-capture and RT-PCR, and developed a quick double-specificity method for detecting and distinguishing different HCV-Ig complexes. We compared our new method, the immuno-capture RT-PCR (iRT-PCR), with the conventional RT-PCR (cRT-PCR) for the sensitivity of detecting HCV in 35 clinically diagnosed patients with HCV infection. The results showed that 31 patients were detected to be positive by using iRT-PCR, whereas 16 patients were positive with the use of cRT-PCR. HCV-IgM, HCV-IgG, HCV-IgA could separately be detected by iRT-PCR and their positive rates were 66.7%, 51.0%, 62.7%, respectively. HCV bound to antibody was a common feature of hepatitis C (HC) and 86.3% of patients were positive at least by one of the HCV-Ig tests. The patterns of HCV RNA constituents varied according to disease categories. In summary, iRT-PCR is a valuable method for analysis of the composition of the immune complexes, which may provide new and valuable insights into HCV pathogenesis.

Adolescent↗

PubMed2001

[Fusion expression of a peptide antibiotic-apidaecin gene in Lactococcus lactis].

The ubiquitin fusion of apidaecin was expressed in Lactococcus lactis, using a novel nisin-inducible expression system. After induction, a specific band could be detected in the extracts of the host strain by Tricine-SDS-PAGE and Western blotting. Production of the fusion was up to 7.2% of the total soluble protein of the host strain. While the fusion was cut by ubiquitin specific protease-UBP1, the product had distinct antibacterial activity.

Amino Acid Sequence↗

PubMed2001

[The primary role of central region of HC-pro of potato Y potyvirus in synergism of plant viruses].

Five deleted mutants of HC-Pro gene of Chinese isolate of potato Y potyvirus (PVY-C) were obtained by PCR mutation, and their plant expression vectors were constructed. They were transformed into tobacco K326 (Nicotina tabacum cv. K326) mediated by Agrobacterium. PCR and Southern blot analysis revealed that PVY-C HC-Pro gene and its deleted mutants were integrated into tobacco genome, and Western blot analysis showed that they were all expressed in transgenic tobacco plants. Furthermore, infection test demonstrated that the central region of PVY-C HC-Pro can mediate synergism of PVY-C/cucumber mosaic cucumovirus (CMV) and PVY-C/potato X potexvirus (PVX), identifying that it is functional domain in synergism.

Blotting, Southern↗

PubMed2001

[Cloning of promoter of banana fruit-specific ACC synthase gene and primary study on its function].

Based on our cDNA sequence of ACC synthase gene expressed specifically in banana fruit, we cloned the 5'-flanking proximal region of 1197 bp and distal region of 1556 bp via walking on unknown region of genomic DNA by an improved adaptor ligation PCR, and created a 2591 bp fragment containing a promoter region of 2505 bp and a transcriptional region of 86 bp. The entire 2.5 kb promoter and 5 different 5' deletion variants were fused to the GUS (beta-glucuronidase) cDNA sequence and introduced into leaf, root and fruit cells of banana plant via particle bombardment separately. The results of transient gene expression assay indicated that the obtained promoter region could direct fruit-specific gene expression. The regulatory region for fruit-specificity was possibly located in the region from -1111 to +1 and a positive regulatory region may locate between nucleotide -1111 to -608.

Base Sequence↗

PubMed2001

[Construction of plant expression vectors harboring a peptide antibiotic-apidaecin gene and resistance analysis of the transgenic tobacco].

Two plant expression vectors(pBinPRHbI and pBinPRSIHbI) were constructed: Firstly, apidaecin gene were fused to the signal peptide coding sequencing of a PR-protein, and cloned into a binary vector pBin438 to form pBinPRHbI. Then, the cassette consisting of 35S promoter, PR signal peptide coding sequencing and apidaecin gene was cut off from pBinPRHbI and inserted into another plant expression vector pBinPRSI to produce a bivalent plant expression vector pBinPRSIHbI. pBinPRSI was constructed previously in our lab and contained PR signal peptide and Shiva-I fusion gene under control of 35S promoter. The three plant expression vectors were introduced into tobacco by Agrobacterium-mediated transformation. The positive rate of PCR was 95% in all putative transgenic plants. Results from Southern blot indicated that foreign genes were integrated into tobacco genome and RT-PCR analysis proved that the foreign gene was transcribed in transgenic tobacco. The transgenic tobacco showed higher resistance to P. syringae pv tabaci, the causal agent of tobacco wild fire disease, than their original cultivars. From the disease index, the transgenic plants carrying apidaecin and Shiva-I genes had highest resistance among three kinds of transgenic plants, and the plants carrying Shiva-I gene alone had lowest resistance.

Anti-Infective Agents↗

PubMed2001

[Cloning of promoter of banana fruit ripening-related ACO1 and primary study on its function].

According to the published sequence of ACC oxidase gene(ACO1) which was shown to express in banana fruits, we cloned its 5'-flanking region by improved adaptor ligation PCR. The 1526 bp fragment contained a putative TATA-box. Sequence alignment of our promoter with two published (934 bp and 1451 bp) revealed 97.3% (López-Gómez et al.) and 88.8% (May and Kipp) similarity in nucleotide sequence, respectively. The 5'-flanking fragments in different length were fused to the coding sequence of GUS gene. These constructs were delivered to leaf, root and fruit cells of banana via particle bombardment. The results of transient gene expression assay showed that the cloned ACO1 promoter was able to direct fruit-specific gene expression, and that the regulatory region for fruit-specificity was probably located in the 0.7 kb 5'-flanking sequence of the gene and some positive regulatory elements may exist within the region of 355 bp from 468 to 822.

Amino Acid Oxidoreductases↗

PubMed2001

[Differentiation of human hepatocarcinoma SMMC-7721 cells induced by HMBA].

In this paper, the effects of HMBA on the differentiation of human hepatocarcinoma cell line SMMC-7721 were investigated. After treated with 5 mmol/L HMBA, the proliferation of SMMC-7721 cells was inhibited remarkably, the cell growth inhibitory rate amounted to 64.14%, the cell mitotic index was declined by 53.88%. Light microscopy and transmission electron microscopy showed that the morphology and ultrastructure of the cells treated with HMBA undergone restorational alteration. Cytochemistry and immunocytochemistry assay revealed that the activities of gamma-GT declined and the levels of AFP and PCNA downregulated while the activity of TAT increased significantly after HMBA treatment. In the meantime, flow cytometry analysis showed that HMBA could arrest the cells in G0/G1 phase. The results showed that HMBA could effectively inhibit the proliferation, reverse the malignant morphology and ultrastructure, alter the levels of enzymes and antigens, arrest the cells in G0/G1, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells in vitro.

Acetamides↗

PubMed2000

[Cloning and sequence of cDNA encoding ACC synthase specifically expressed in banana fruit].

A cDNA encoding ACC synthase in banana pulp was amplified by RT-PCR and cloned in E. coli. The 5' terminal region of the ACC synthase transcript was determined by using 5' RACE procedure. The results showed that the ACC synthase cDNA in banana pulp is 1752 bp in length including 74 bp of 5' untranslated region, 1461 bp of coding region which encodes a polypeptide of 486 amino acides and 217 bp of 3' untranslated region. The Northern blot analysis indicates that the ACC synthase mRNA is specifically transcripted in banana fruits.

Blotting, Northern↗

PubMed1999

Protection of sinusoidal endothelial cells against storage/reperfusion injury by prostaglandin E2 derived from Kupffer cells.

BACKGROUND: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats. METHODS: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections. RESULTS: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants. CONCLUSION: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.

Animals↗

PubMed1999

A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.

In hepatocellular carcinoma (HCC), autoantibodies to intracellular antigens are detected in 30-40% of patients. Patients with chronic hepatitis or liver cirrhosis develop HCC, and when this occurs, some patients exhibit autoantibodies of new specificities. It has been suggested that these novel autoantibody responses may be immune system reactions to proteins involved in transformation-associated cellular events. One HCC serum shown to contain antibodies to unidentified cellular antigens was used to immunoscreen a cDNA expression library, and a full length cDNA clone was isolated with an open reading frame encoding 556 amino acids with a predicted molecular mass of 62 kD. The 62-kD protein contained two types of RNA-binding motifs, the consensus sequence RNA-binding domain (CS-RBD) and four hnRNP K homology (KH) domains. This protein, provisionally called p62, has close identity (66-70%) to three other proteins at the amino acid sequence level, and all four proteins may belong to a family having CS-RBD in the NH2-terminal region and four KH domains in the mid-to-COOH- terminal region. The homologous proteins are: KH domain-containing protein overexpressed in cancer (Koc); zipcode binding protein, a protein which binds to a conserved nucleotide element in chicken beta-actin mRNA (ZBP1); and a protein which binds to a promoter cis element in Xenopus laevis TFIIIA gene (B3). p62 protein is cytoplasmic in location, and autoantibodies were found in 21% of a cohort of HCC patients. Patients with chronic hepatitis and liver cirrhosis, conditions which are frequent precursors to HCC, were negative for these autoantibodies, suggesting that the immune response might be related to cellular events leading to transformation. However, the possible involvement of p62 autoantigen as a factor in the transformation process remains to be elucidated.

Amino Acid Sequence↗

PubMed1999

[Stable expression of recombinant inducible nitric oxide synthase in NG108-15 cells and its biological characterization].

The neuroblastoma x glioma NG108-15 cells were transfected with recombinant eukarytic expression plasmid pCMViNOS containing the full-length cDNA encoding inducible nitric oxide synthase (iNOS). A lot of G418-resistant clones were screened at 600 micrograms/ml of geneticin. In the 2# clone expressing iNOS gene, iNOS catalytic activity in the cytosol fraction displayed to have an increasing trend, accompanying with the accumulation of NO2- content in the supernantant of cultured cells and the intracellular cGMP concentration, which suggested that NO-cGMP signal pathway was mediated by the expression of iNOS gene and blocked by NG-nitro-L-arginine (L-NNA) and methylene blue (MB). Activity of iNOS was concentration-dependently inhibited by NOS inhibitors such as L-NNA and aminoguanidine. The result of measurement of NADPH diaphorase activity and immunocytochemical staining showed that localization of the function expression of iNOS protein mainly existed in the cytoplasm of NG108-15 cells transfected with pCMViNOS. Furthermore, the chromosomal integration, transcript and protein translation of foreign iNOS gene were identified by Southern hybridization, RT-PCR and Western blot, respectively. The results indicated that iNOS gene-transfected cells had mRNA transcription and specific protein expression at high level. Given the above results, the engineering cell line with stable expression of iNOS gene was successfully established. The new neuronal cell line may serve as a source of iNOS and provide a useful cell model for studying iNOS biological function and developing novel iNOS-selective inhibitors.

Animals↗

PubMed1998

Construction of an inducible nitric-oxide synthase gene transferring vector mediated by retrovirus.

AIM: To construct an inducible nitric-oxide synthase (iNOS) gene transferring vector mediated by retrovirus. METHODS: Recombinant DNA and polymerase chain reaction (PCR) amplification techniques were used. RESULTS: With 2 steps of molecular cloning, the full-length cDNA encoding macrophage iNOS was isolated from plasmid pKSiNOS and subcloned into intermediate vector pSP72, adjusting the restriction enzyme sites in both 5'- and 3'-flanking ends of insert fragment. The retroviral vector pLNCXiNOS which contains iNOS coding region, cytomegalovirus promoter and neomycin resistance (neor) gene was further constructed. The authenticity of insertion size and orientation of iNOS sequence was verified by restriction mapping and PCR analysis with iNOS gene-specific primers. CONCLUSION: Retroviral expression vector carrying iNOS fragment is obtained, which provides a material to establish a model of iNOS gene-modified neurons.

Cloning, Molecular↗